scholarly journals Whole-genome fetal and maternal DNA methylation analysis using MeDIP-NGS for the identification of differentially methylated regions

2016 ◽  
Vol 98 ◽  
Author(s):  
ANNA KERAVNOU ◽  
MARIOS IOANNIDES ◽  
KYRIAKOS TSANGARAS ◽  
CHARALAMBOS LOIZIDES ◽  
MICHAEL D. HADJIDANIEL ◽  
...  
Keyword(s):  
Dna Methylation ◽  
Chorionic Villus ◽  
Maternal Blood ◽  
Maternal Plasma ◽  
Chorionic Villus Sampling ◽  
Whole Genome ◽  
Differentially Methylated Regions ◽  
Epigenetic Marker ◽  
Novel Approach ◽  
Targeted Enrichment

SummaryDNA methylation is an epigenetic marker that has been shown to vary significantly across different tissues. Taking advantage of the methylation differences between placenta-derived cell-free DNA and maternal blood, several groups employed different approaches for the discovery of fetal-specific biomarkers. The aim of this study was to analyse whole-genome fetal and maternal methylomes in order to identify and confirm the presence of differentially methylated regions (DMRs). We have initially utilized methylated DNA immunoprecipitation (MeDIP) and next-generation sequencing (NGS) to identify genome-wide DMRs between chorionic villus sampling (CVS) and female non-pregnant plasma (PL) and peripheral blood (WBF) samples. Next, using specific criteria, 331 fetal-specific DMRs were selected and confirmed in eight CVS, eight WBF and eight PL samples by combining MeDIP and in-solution targeted enrichment followed by NGS. Results showed higher enrichment in CVS samples as compared to both WBF and PL samples, confirming the distinct methylation levels between fetal and maternal DNA for the selected DMRs. We have successfully implemented a novel approach for the discovery and confirmation of a significant number of fetal-specific DMRs by combining for the first time MeDIP and in-solution targeted enrichment followed by NGS. The implementation of this double-enrichment approach is highly efficient and enables the detailed analysis of multiple DMRs by targeted NGS. Also, this is, to our knowledge, the first reported application of MeDIP on plasma samples, which leverages the implementation of our enrichment methodology in the detection of fetal abnormalities in maternal plasma.

Chorionic villus sampling increases the alpha-fetoprotein level but not the number of fetal erythrocytes in maternal blood

2004 ◽  
Vol 191 (6) ◽  
pp. S72
Author(s):  
Denise Pelikan ◽  
Sicco Scherjon ◽  
Wilma Mesker ◽  
Godelieve Groot-Swings ◽  
Hans Tanke ◽  
...  
Keyword(s):  
Chorionic Villus ◽  
Maternal Blood ◽  
Alpha Fetoprotein ◽  
Chorionic Villus Sampling

scholarly journals Large-scale transcriptome-wide profiling of microRNAs in human placenta and maternal plasma at early to mid gestation

2020 ◽  
Author(s):  
Melanie D. Smith ◽  
Katherine Pillman ◽  
Tanja Jankovic-Karasoulos ◽  
Dale McAninch ◽  
Qianhui Wan ◽  
...  
Keyword(s):  
Gestational Age ◽  
Human Placenta ◽  
Large Scale ◽  
Chorionic Villus ◽  
Maternal Blood ◽  
Maternal Plasma ◽  
Placental Development ◽  
Early Gestation ◽  
Oxygen Environment ◽  
Mirna Clusters

AbstractBackgroundMicroRNAs (miRNAs) are increasingly seen as important regulators of placental development and opportunistic biomarker targets. Given the difficulty in obtaining samples from early gestation and subsequent paucity of the same, investigation of the role of miRNAs in early gestation human placenta has been limited. To address this, we generated miRNA profiles using 96 placentas from presumed normal pregnancies, across early gestation, in combination with matched profiles from maternal plasma. Placenta samples range from 6-23 weeks’ gestation, a time period that includes placenta from the early, relatively low but physiological (6-10 weeks’ gestation) oxygen environment, and later, physiologically normal oxygen environment (11-23 weeks’ gestation).ResultsWe identified 637 miRNAs with expression in 86 samples (after removing poor quality samples), showing a clear gestational age gradient from 6-23 weeks’ gestation. We identified 374 differentially expressed (DE) miRNAs between placentas from 6-10 weeks’ versus 11-23 weeks’ gestation. We see a clear gestational age group bias in miRNA clusters C19MC, C14MC, miR-17∼92 and paralogs, regions that also include many DE miRNAs. Proportional change in expression of placenta-specific miRNA clusters was reflected in maternal plasma.ConclusionThe presumed introduction of oxygenated maternal blood into the placenta (between ∼10-12 weeks’ gestation) changes the miRNA profile of the chorionic villus, particularly in placenta-specific miRNA clusters. Data presented here comprise a clinically important reference set for studying early placenta development and may underpin the generation of minimally invasive methods for monitoring placental health.

1 Whole-genome bisulfite sequencing of bovine gametes and invivo-produced pre-implantation embryos

2019 ◽  
Vol 31 (1) ◽  
pp. 126
Author(s):  
J. E. Duan ◽  
Z. Jiang ◽  
F. Alqahtani ◽  
I. Mandoiu ◽  
H. Dong ◽  
...  
Keyword(s):  
Dna Methylation ◽  
Single Cell ◽  
Epigenetic Regulation ◽  
Bisulfite Sequencing ◽  
Whole Genome ◽  
Differentially Methylated Regions ◽  
Whole Genome Bisulfite Sequencing ◽  
Cpg Sites ◽  
Genome Wide ◽  
Genome Bisulfite Sequencing

Dynamic changes in DNA methylation are crucial in the epigenetic regulation of mammalian embryogenesis. Global DNA methylation studies in the bovine, however, remain mostly at the immunostaining level. We adopted the single-cell whole-genome bisulfite sequencing method to characterise stage-specific genome-wide DNA methylation in bovine sperm, individual oocytes derived invivo and invitro, and invivo-developed embryos at the 2-, 4-, 8-, and 16-cell stages. This method allowed us to theoretically cover all CpG sites in the genome using a limited number of cells from single embryos. Pools of 20 sperm were selected from a bull with proven fertility. Single oocytes (n=6) and embryos (n=4 per stage) were collected from Holstein cows (n=10). Single-cell whole-genome bisulfite sequencing libraries were prepared and sequenced using the Illumina HiSEqn 4000 platform (Illumina, San Diego, CA, USA). Sequencing reads were filtered and aligned to the bovine reference genome (UMD 3.1.1) using Bismark (Krueger and Andrews 2011Bioinformatics27, 1571-1572, DOI: 10.1093/bioinformatics/btr167).A 300-bp tile-based method was applied to bin the genome into consecutive windows to facilitate comparison across samples. The DNA methylation level was calculated as the fraction of read counts of the total number of cytosines (methylated) in the total read counts of reported cytosines and thymines (methylated and unmethylated), only if more than 3 CpG sites were covered in this tile. Gamete-specific differentially methylated regions were identified when DNA methylation levels were greater than 75% in one type of gamete and less than 25% in the other with false discovery rate-corrected Fisher’s exact test P-values of less than 0.05. The major wave of genome-wide DNA demethylation was complete at the 8-cell stage when de novo methylation became prominent. Sperm and oocytes had numerous differentially methylated regions that were enriched in intergenic regions. Differentially methylated regions were also identified between invivo- and invitro-matured oocytes. Moreover, X chromosome methylation followed the global dynamic patterns. Virtually no (less than 1.5%) DNA methylation was found in mitochondrial DNA. Finally, using our RNA sequencing data generated from the same developmental stages (Jiang et al. 2014 BMC Genomics 15, 756; DOI: 10.1186/1471-2164-15-756), we revealed an inverse correlation between gene expression and promoter methylation. Our study provides the first fully comprehensive analysis of the global dynamics of DNA methylation in bovine gametes and single early embryos using single-cell whole-genome bisulfite sequencing. These data provide insights into the critical features of the methylome of bovine embryos and serve as an important reference for embryos produced by assisted reproduction, such as IVF and cloning, and a model for human early embryo epigenetic regulation.

scholarly journals Noninvasive Prenatal Diagnosis from Maternal Blood: Finally Available after 20 Years of Research

2013 ◽  
Vol 7 (4) ◽  
pp. 440-442
Author(s):  
Wolfgang Holzgreve
Keyword(s):  
Prenatal Diagnosis ◽  
Single Gene ◽  
Chorionic Villus ◽  
Maternal Blood ◽  
Chorionic Villus Sampling ◽  
Clinical Use ◽  
Maternal Circulation ◽  
Isolated Cells ◽  
Noninvasive Prenatal Diagnosis ◽  
Cell Free Fetal Dna

ABSTRACT Since all prenatal invasive procedures, such as amniocentesis and chorionic villus sampling carry a small risk for the pregnant woman and a risk to induce the loss of a pregnancy of up to 1%, there have been efforts now for at least a quarter of a century to develop a noninvasive method from the blood of pregnant women. First there was a considerable effort to isolate fetal cells from maternal circulation, and these techniques were carefully evaluated in a NIH-sponsored study of a few US American centers and ours in Basel/Switzerland. It turned out; however, that interphase fluorescence to identify fetal aneuploidies from these isolated cells was not reliable enough for clinical use. The breakthrough came with the recognition of the group by D Lo et al; who showed for the first time that cell-free fetal DNA in maternal plasma and serum can be used reliably for prenatal diagnosis. One of the first successful applications was the detection of the fetal Rhesus factor around 11 weeks of gestation in pregnancies of Rhesus-negative mothers. The Sequenom Company in San Diego, USA, which acquired the patent of D Lo et al on the use of cell free DNA and ours on size separation of fetal vs maternal DNA subsequently showed in large series that the noninvasive prenatal diagnosis of fetal trisomy 21 from maternal blood by massive parallel sequencing has an accuracy around 99%, and currently up to 100,000 cases have been investigated already in different laboratories. Also the noninvasive prenatal diagnosis of trisomies 18 and 13 is possible, and an increasing amount of single gene anomalies will be diagnosable in the future noninvasively. The whole development of noninvasive prenatal diagnosis is appositive example that long-term research pays-off to bring a concept from the first steps finally into clinical use. How to cite this article Holzgreve W. Noninvasive Prenatal Diagnosis from Maternal Blood: Finally Available after 20 Years of Research. Donald School J Ultrasound Obstet Gynecol 2013;7(4):440-442.

scholarly journals Prenatal Diagnosis, Where and How: No Way Out?

2014 ◽  
Vol 8 (3) ◽  
pp. 293-310
Author(s):  
Franco Borruto ◽  
Alain Treisser ◽  
Ciro Comparetto
Keyword(s):  
Down Syndrome ◽  
Prenatal Diagnosis ◽  
Amniotic Fluid ◽  
Diagnostic Tests ◽  
Genetic Diseases ◽  
Chorionic Villus ◽  
Maternal Blood ◽  
Screening Tests ◽  
Chorionic Villus Sampling ◽  
Second Trimester

ABSTRACT Prenatal diagnosis is the branch of medicine and in particular of obstetrics, that studies and applies the techniques that reveal the normality or the presence of diseases of various kinds, in the fetus. All the techniques of prenatal diagnosis are performed during pregnancy and may be invasive or less. Among the best known, amniocentesis is the most exploited technique nowadays to highlight the possible presence of chromosomal disorders in the fetus, but also infections and genetic diseases such as thalassemia, cystic fibrosis, hemophilia, spina bifida, albinism. Amniocentesis consists of taking an amniotic fluid sample which is then analyzed. Fetal cells suspended in the withdrawn liquid allow us to reconstruct the chromosome map of the fetus and then to confirm or not its normality. Genetic testing, however, are not able to recognize the physical or mental characteristics of the unborn child which are the result of the interaction between multiple genes and the environment. Amniotic fluid makes possible to perform other types of analysis, more or less complex, and it is also possible to store the amniotic stem cells. Similar to amniocentesis as a principle but different as a technique, is chorionic villus sampling (CVS), in which the cells can be put in culture to show their normality, but they are cells taken outside from the gestational chamber (chorionic villi). These are invasive techniques (the fluid is taken by puncture in both cases), but there are also noninvasive techniques. The development of ultrasound, for example, has made it possible to develop some highly sensitive diagnostic techniques, such as the first trimester combined test [bitest and nuchal translucency (NT)], the ‘quadruple’ test, and lately the SCA test in the second trimester, all based on the ultrasound measurement of anatomical and functional parameters of the fetus and on the results of blood tests. These are all screening tests, then they do not give a definite answer but they have a statistical value (very accurate) that can direct toward diagnostic tests. Recently, an extremely sensitive test for the most common aneuploidies and in particular Down syndrome has been proposed to be performed on maternal blood. This test (called fetal DNA testing) is based on the count of fragments of specific chromosomes (21 in the case of Down syndrome) in maternal blood. Although not belonging to diagnostic tests but to probabilistic ones, this test is absolutely the most accurate so far available, with values around 99.99% sensitivity and 0.2% false positives. Also ultrasound in the second trimester of pregnancy (also called morphological ultrasound) that can detect any malformation or fetal abnormality and fetal echocardiography, which analyzes sonographically the fetal heart not only anatomically but also from the dynamic-functional point of view, may be considered methods of prenatal diagnosis. This technique cannot identify genetic diseases. How to cite this article Borruto F, Treisser A, Comparetto C. Prenatal Diagnosis, Where and How: No Way Out? Donald School J Ultrasound Obstet Gynecol 2014;8(3):293-310.

FETAL NUCLEIC ACIDS IN MATERNAL PLASMA

2006 ◽  
Vol 17 (2) ◽  
pp. 125-137
Author(s):  
TRACY YH LEE ◽  
YM DENNIS LO
Keyword(s):  
Prenatal Diagnosis ◽  
Pregnant Women ◽  
Chorionic Villus ◽  
Fetal Loss ◽  
Maternal Plasma ◽  
Chorionic Villus Sampling ◽  
Invasive Procedures ◽  
Noninvasive Prenatal Diagnosis ◽  
The World ◽  
Definitive Methods

Prenatal diagnosis is now an established part of modern obstetrical practice around the world. While the current definitive methods for prenatal diagnosis rely mainly on invasive procedures such as chorionic villus sampling and amniocentesis, such procedures carry a low but definite risk of fetal loss. As a consequence of the procedure-associated risk of miscarriage, prenatal diagnosis is currently limited to pregnant women with an increased likelihood of bearing an abnormal fetus. To extend the application of prenatal diagnosis to all pregnant women, it has been a long-sought goal of researchers worldwide to introduce safer methods for prenatal diagnosis, towards noninvasive prenatal diagnosis.

scholarly journals Non invasive prenatal diagnosis of ?- Thalassemia, A narrative review study

2017 ◽  
Vol 16 (2) ◽  
pp. 196-202 ◽  
Author(s):  
Mandana Zafari ◽  
Mehrnoush Kowsaryan ◽  
Pooria Gill ◽  
Ali Banihashemi
Keyword(s):  
Prenatal Diagnosis ◽  
Medical Science ◽  
Chorionic Villus ◽  
Maternal Plasma ◽  
Paternal Allele ◽  
Chorionic Villus Sampling ◽  
Non Invasive ◽  
Invasive Method ◽  
Review Study ◽  
Chromosomal Aneuploidies

?- Thalassemia is major monogenic disorder. A practical way to prevention of Thalassemia is identification of carries couples; genetic counseling and offer prenatal diagnose services for both carrier couples. Routine prenatal diagnose are chorionic villus sampling and amniocentesis, but both of them are invasive method and they can be ended to bleeding and pregnancy loss. Recently non invasive prenatal diagnosis has been done by researchers for early detection of pre-eclampsia, chromosomal aneuploidies, RhD-genotyping. Regarding non invasive prenatal diagnosis of ?- Thalassemia, detection of paternally inherited mutation in maternal plasma is possible. If the fetus inherited normal paternal allele the performance of invasive method it is not necessary, so this method can be eliminate 50% performance of routine prenatal diagnosis.Bangladesh Journal of Medical Science Vol.16(2) 2017 p.196-202

scholarly journals Systematic Search for Placental DNA-Methylation Markers on Chromosome 21: Toward a Maternal Plasma-Based Epigenetic Test for Fetal Trisomy 21

2008 ◽  
Vol 54 (3) ◽  
pp. 500-511 ◽  
Author(s):  
Stephen S C Chim ◽  
Shengnan Jin ◽  
Tracy Y H Lee ◽  
Fiona M F Lun ◽  
Wing S Lee ◽  
...  
Keyword(s):  
Dna Methylation ◽  
Blood Cells ◽  
Trisomy 21 ◽  
Maternal Blood ◽  
Maternal Plasma ◽  
Chromosome 21 ◽  
Noninvasive Detection ◽  
Fetal Dna ◽  
Noninvasive Prenatal Diagnosis ◽  
Methylation Patterns

Abstract Background: The presence of fetal DNA in maternal plasma represents a source of fetal genetic material for noninvasive prenatal diagnosis; however, the coexisting background maternal DNA complicates the analysis of aneuploidy in such fetal DNA. Recently, the SERPINB5 gene on chromosome 18 was shown to exhibit different DNA-methylation patterns in the placenta and maternal blood cells, and the allelic ratio for placenta-derived hypomethylated SERPINB5 in maternal plasma was further shown to be useful for noninvasive detection of fetal trisomy 18. Methods: To develop a similar method for the noninvasive detection of trisomy 21, we used methylation-sensitive single nucleotide primer extension and/or bisulfite sequencing to systematically search 114 CpG islands (CGIs)—76% of the 149 CGIs on chromosome 21 identified by bioinformatic criteria—for differentially methylated DNA patterns. The methylation index (MI) of a CpG site was estimated as the proportion of molecules methylated at that site. Results: We identified 22 CGIs which were shown to contain CpG sites that were either completely unmethylated (MI = 0.00) in maternal blood cells and methylated in the placenta (MI range, 0.22–0.65), or completely methylated (MI = 1.00) in maternal blood cells and hypomethylated in the placenta (MI range, 0.00–0.75). We detected, for the first time, placental DNA-methylation patterns on chromosome 21 in maternal plasma during pregnancy and observed their postpartum clearance. Conclusion: Twenty-two (19%) of the 114 studied CGIs on chromosome 21 showed epigenetic differences between samples of placenta and maternal blood cells; these CGIs may provide a rich source of markers for noninvasive prenatal diagnosis.

scholarly journals Whole Genome Bisulfite Sequencing of Purified Mouse Promyelocytes Reveals Differentially Methylated Regions in Cells Expressing PML-Rara

Blood ◽  
2014 ◽  
Vol 124 (21) ◽  
pp. 3531-3531
Author(s):  
Shamika Ketkar-Kulkarni ◽  
Christopher B Cole ◽  
David H. Spencer ◽  
Angela M. Verdoni ◽  
Nichole Havey ◽  
...  
Keyword(s):  
Gene Expression ◽  
Dna Methylation ◽  
Latent Period ◽  
Bisulfite Sequencing ◽  
De Novo ◽  
Chromosome 8 ◽  
Whole Genome ◽  
Chromosome 4 ◽  
Differentially Methylated Regions ◽  
Genome Bisulfite Sequencing

Abstract Acute promyelocytic leukemia (APL) is an AML subtype that is characterized by aberrant expansion of immature myeloid progenitors and precursors that are arrested at the promyelocyte stage. Almost all APL cases are characterized by the t(15;17)(q22;q11.2) translocation that creates the PML-RARA fusion oncogene. Human APL cells are known to have a canonical expression signature and a specific methylation phenotype that is unique to this form of AML. Our laboratory previously created a mouse model of APL by expressing a human PML-RARA cDNA from the mouse Cathepsin G (Ctsg) locus (Ctsg-PML-RARA), which activates human PML- RARA expression in early myeloid progenitor cells, with peak expression in promyelocytes. After a long latent period (6-12 months), ~60% of these mice develop a clonal, APL-like myeloid malignancy. The long latent period is probably due to the requirement for cooperating mutations that synergize with PML-RARA to accelerate the disease. Human APL samples have a unique gene expression signature that distinguishes them from all other subtypes of AML. We evaluated RNA-Seq data derived from Poly A+ enriched cDNAs obtained from purified promyelocytes derived from 3 young (6 week old) WT and 3 Ctsg-PML-RARA mice. We identified 779 annotated genes that are significantly dysregulated in murine promyelocytes expressing PML-RARA with a log2 fold change >= 2 and P<0.05. Some of these genes included Spib/Pu.1, Pou2af1, Jak2, Runx1, and many others. We also identified a set of 24,018 RNAs in promyelocytes that were defined as novel transcripts. This set contains 7,413 lncRNAs with an FPKM value of >= 2. Differential expression analysis yielded 56 dysregulated lncRNA regions in PML-RARA expressing promyelocytes. To explore the association between gene dysregulation and DNA methylation in promyelocytes, we carried out whole-genome bisulfite sequencing using DNA derived from the purified promyelocytes of a 6 week old Ctsg-PML-RARA mouse, and a WT littermate. We generated a total of approximately 800 million sequencing reads, of which 78% mapped uniquely to the reference genome (mm9); we were able to map ~19 million CpGs with at least 10x coverage. Differential methylation analysis performed on ~4.5 million 1 Kb windows spanning the entire genome identified 17,633 differentially methylated regions with a mean difference of >= 25% and a q-value of < 0.01, the vast majority of which (17,264, 98%) were hypomethylated in the Ctsg-PML-RARA promyelocytes. These windows overlap several known genes, including Runx1, Jak2, Dnmt3a, Gata2, and the Hoxa and Hoxb gene clusters. Using more strict criteria (> 50% mean methylation difference), we identified 87 differentially methylated regions of at least 2 Kb in size. Of these 87 distinct regions, 74 (85%) were hypomethylated in PML-RARA promyelocytes, and 13 were hypermethylated; examples of both as shown in Figure 1. These data strongly suggest that PML-RARA has at least two distinct mechanisms by which it can modify DNA methylation. In regions where CpGs are hypomethylated, PML-RARA may be blocking the normal methylation of CpGs by the de novo DNA methyltransferases Dnmt3a and/or Dnmt3b. In contrast, PML-RARA may be directing de novo methyltransferases to act on the hypermethylated regions. Regardless, these data, when coupled with comprehensive chromatin accessibility mapping and complete RNA sequencing data, should provide new insights into the mechanisms used by PML-RARA to alter gene expression and initiate APL. Figure1. Examples of differentially methylated regions. Black=WT cells. Red=PML-RARA expressing cells. Each CpG in the region is represented as a dot. Scale is 0-100% methylated at each position. Top panel: a region on chromosome 8 that is hypomethylated in PML-RARA expressing promyelocytes. Bottom panel: a region on chromosome 4 that is hypermethylated in PML-RARA expressing promyelocytes. Figure1. Examples of differentially methylated regions. Black=WT cells. Red=PML-RARA expressing cells. Each CpG in the region is represented as a dot. Scale is 0-100% methylated at each position. Top panel: a region on chromosome 8 that is hypomethylated in PML-RARA expressing promyelocytes. Bottom panel: a region on chromosome 4 that is hypermethylated in PML-RARA expressing promyelocytes. Disclosures No relevant conflicts of interest to declare.

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