Identification of fetal unmodified and 5-hydroxymethylated CG sites in maternal cell-free DNA for non-invasive prenatal testing

Gordevičius, Juozas; Narmontė, Milda; Gibas, Povilas; Kvederavičiūtė, Kotryna; Tomkutė, Vita; Paluoja, Priit; Krjutškov, Kaarel; Salumets, Andres; Kriukienė, Edita

doi:10.1186/s13148-020-00938-x

Cited by 14 publications

(12 citation statements)

References 50 publications

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“…While the DNA that was sonicated for 4 min (4 m.s cffDNA) broke into fragments that ranged from 100 to 1000 bp. As it has also been shown in utero that DNA fragments released from cells via apoptosis or necrosis are often hypomethylated ( Gordevičius et al, 2020 ), we assessed the methylation status of the cffDNA. The methylation ranged from 0.1% to 0.4% ( Figure 1B ) but this did not decrease after 4 min of sonication as no statistical difference in the percentages of methylation was seen when the DNA was sheared into smaller fragments (variation between the samples before and after sonication ± 5%–10%, which was within the range of assay variation for technical repeat samples).…”

Section: Resultsmentioning

confidence: 99%

Fetal DNA Causes Sex-Specific Inflammation From Human Fetal Membranes

Saito-Reis

Kurashima

et al. 2022

Front. Physiol.

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Inflammation is central to the mechanisms of parturition, but the lack of understanding of how it is controlled in normal parturition hampers our ability to understand how it may diverge resulting in preterm birth. Cell-free fetal DNA is found in the amniotic fluid, and it is thought to be able to activate inflammation as a danger-associated molecular pattern. Although its levels increases with gestational age, its effect has not been studied on the human fetal membranes. Thus, the aim of this study was to determine if the fetal DNA can trigger inflammation in the human fetal membranes and, thus, potentially contribute to the inflammatory load. Isolated human amniotic epithelial cells and fetal membrane explants were treated apically with fetal DNA causing the translocation of NF-KB into the nucleus of cells and throughout the cells of the explant layers with time. Fetal membrane explants were treated apically with either small or larger fragments of fetal DNA. IL-6, TNFα, and GM-CSF secretion was measured by ELISA, and pro-MMP2 and pro-MMP9 activity was measured by zymography from apical and basal media. Increased apical IL-6 secretion and basal pro-MMP2 activity was seen with small fragments of fetal DNA. When the data were disaggregated based on fetal sex, males had significant increases in IL-6 secretion and basal increased activity in pro-MMP2 and 9, whereas females had significantly increased basal secretion of TNFα. This was caused by the smaller fragments of fetal DNA, whereas the larger fragments did not cause any significant increases. Male fetal DNA had significantly lower percentages of methylation than females. Thus, when the cytokine and pro-MMP activity data were correlated with methylation percentage, IL-6 secretion significantly correlated negatively, whereas GM-CSF secretion positively correlated. These data support the role of fetal DNA as an inflammatory stimulus in the FM, as measured by increased NF-κB translocation, cytokine secretion, and increased pro-MMP activity. However, the data also suggested that the responses are different from FM tissues of male and female fetuses, and both the fragment size and methylation status of the fetal DNA can influence the magnitude and type of molecule secreted.

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Section: Resultsmentioning

confidence: 99%

Fetal DNA Causes Sex-Specific Inflammation From Human Fetal Membranes

Saito-Reis

Kurashima

et al. 2022

Front. Physiol.

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“…To compare the distribution of oxi-mCGs and their predecessor 5mCGs across genes and TEs, we used our strategy to calculate modified CG fractions, as described previously [ 61 , 62 ]. Importantly, for WGBS data, the calculated 5mCG fractions across genes correlated well with 5mCG methylation values (0.94 for C. cinerea and 0.54 for L. bicolor ).…”

Section: Resultsmentioning

confidence: 99%

Distribution and regulatory roles of oxidized 5-methylcytosines in DNA and RNA of the basidiomycete fungi Laccaria bicolor and Coprinopsis cinerea

et al. 2022

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The formation of three oxidative DNA 5-methylcytosine (5mC) modifications (oxi-mCs)—5-hydroxymethylcytosine (5hmC), 5-formylcytosine (5fC) and 5-carboxylcytosine (5caC)—by the TET/JBP family of dioxygenases prompted intensive studies of their functional roles in mammalian cells. However, the functional interplay of these less abundant modified nucleotides in other eukaryotic lineages remains poorly understood. We carried out a systematic study of the content and distribution of oxi-mCs in the DNA and RNA of the basidiomycetes Laccaria bicolor and Coprinopsis cinerea, which are established models to study DNA methylation and developmental and symbiotic processes. Quantitative liquid chromatography–tandem mass spectrometry revealed persistent but uneven occurrences of 5hmC, 5fC and 5caC in the DNA and RNA of the two organisms, which could be upregulated by vitamin C. 5caC in RNA (5carC) was predominantly found in non-ribosomal RNA, which potentially includes non-coding, messenger and small RNA species. Genome-wide mapping of 5hmC and 5fC using the single CG analysis techniques hmTOP-seq and foTOP-seq pointed at involvement of oxi-mCs in the regulation of gene expression and silencing of transposable elements. The implicated diverse roles of 5mC and oxi-mCs in the two fungi highlight the epigenetic importance of the latter modifications, which are often neglected in standard whole-genome bisulfite analyses.

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“…In der Biotechnologie finden DNA-MTasen zunehmende Bedeutung in der (optischen) Kartierung von DNA, Diagnostik und der DNA-(Hydroxy)-Methylierungsdetektion durch (hm)TOP-seq [19][20][21][22][23][24][25]. Dies ist maßgeblich darauf zuru ¨ckzufu ¨hren, dass auch nicht-natu ¨rliche funktionelle Gruppen sequenzspezifisch auf DNA u ¨bertragen werden ko ¨nnen.…”

Section: Adomet Und Synthetische Analoga Fu ¨R Biotechnologische Anwe...unclassified

DNA‐Methyltransferasen und AdoMet‐Analoga als Werkzeuge für die Molekularbiologie und Biotechnologie

Cornelissen

Hoffmann

Rentmeister

2023

Chemie Ingenieur Technik

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DNA-Methyltransferasen waren lange Zeit vor allem fu ¨r die molekularbiologische Grundlagenforschung interessant, da sie sowohl in Pro-als auch in Eukaryoten wichtige Prozesse regulieren. Seit der Entwicklung von synthetischen Analoga des Cofaktors S-Adenosyl-L-methionin, ist es mo ¨glich, nicht nur Methylgruppen, sondern auch zahlreiche funktionelle Gruppen sequenzspezifisch auf DNA zu u ¨bertragen. Einige DNA-Methyltransferasen weisen aufgrund ihrer Struktur eine bemerkenswerte Promiskuita ¨t bezu ¨glich des Cofaktors auf, so dass die Markierung von DNA mit guter Effizienz gelingen kann. In diesem U ¨bersichtsartikel stellen wir die wichtigen Entwicklungen in diesem Bereich zusammen und diskutieren interessante aktuelle und potentielle Anwendungsmo ¨glichkeiten in der Molekularbiologie und Biotechnologie.

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Identification of fetal unmodified and 5-hydroxymethylated CG sites in maternal cell-free DNA for non-invasive prenatal testing

Cited by 14 publications

References 50 publications

Fetal DNA Causes Sex-Specific Inflammation From Human Fetal Membranes

Fetal DNA Causes Sex-Specific Inflammation From Human Fetal Membranes

Distribution and regulatory roles of oxidized 5-methylcytosines in DNA and RNA of the basidiomycete fungi Laccaria bicolor and Coprinopsis cinerea

DNA‐Methyltransferasen und AdoMet‐Analoga als Werkzeuge für die Molekularbiologie und Biotechnologie

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