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

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Gordevičius , J , Narmontė , M , Gibas , P , Kvederavičiūtė , K , Tomkutė , V , Paluoja , P , Krjutškov , K , Salumets , A & Kriukienė , E 2020 , ' Identification of fetal unmodified and 5-hydroxymethylated CG sites in maternal cell-free DNA for non-invasive prenatal testing ' , Clinical epigenetics , vol. 12 , no. 1 , 153 . https://doi.org/10.1186/s13148-020-00938-x

Title: Identification of fetal unmodified and 5-hydroxymethylated CG sites in maternal cell-free DNA for non-invasive prenatal testing
Author: Gordevičius, Juozas; Narmontė, Milda; Gibas, Povilas; Kvederavičiūtė, Kotryna; Tomkutė, Vita; Paluoja, Priit; Krjutškov, Kaarel; Salumets, Andres; Kriukienė, Edita
Contributor: University of Helsinki, University of Helsinki
University of Helsinki, HUS Gynecology and Obstetrics
Date: 2020-10-20
Language: eng
Number of pages: 14
Belongs to series: Clinical epigenetics
ISSN: 1868-7083
URI: http://hdl.handle.net/10138/321950
Abstract: BackgroundMassively parallel sequencing of maternal cell-free DNA (cfDNA) is widely used to test fetal genetic abnormalities in non-invasive prenatal testing (NIPT). However, sequencing-based approaches are still of high cost. Building upon previous knowledge that placenta, the main source of fetal circulating DNA, is hypomethylated in comparison to maternal tissue counterparts of cfDNA, we propose that targeting either unmodified or 5-hydroxymethylated CG sites specifically enriches fetal genetic material and reduces numbers of required analytical sequencing reads thereby decreasing cost of a test.MethodsWe employed uTOPseq and hmTOP-seq approaches which combine covalent derivatization of unmodified or hydroxymethylated CG sites, respectively, with next generation sequencing, or quantitative real-time PCR.ResultsWe detected increased 5-hydroxymethylcytosine (5hmC) levels in fetal chorionic villi (CV) tissue samples as compared with peripheral blood. Using our previously developed uTOP-seq and hmTOP-seq approaches we obtained whole-genome uCG and 5hmCG maps of 10 CV tissue and 38 cfDNA samples in total. Our results indicated that, in contrast to conventional whole genome sequencing, such epigenomic analysis highly specifically enriches fetal DNA fragments from maternal cfDNA. While both our approaches yielded 100% accuracy in detecting Down syndrome in fetuses, hmTOP-seq maintained such accuracy at ultra-low sequencing depths using only one million reads. We identified 2164 and 1589 placenta-specific differentially modified and 5-hydroxymethylated regions, respectively, in chromosome 21, as well as 3490 and 2002 Down syndrome-specific differentially modified and 5-hydroxymethylated regions, respectively, that can be used as biomarkers for identification of Down syndrome or other epigenetic diseases of a fetus.ConclusionsuTOP-seq and hmTOP-seq approaches provide a cost-efficient and sensitive epigenetic analysis of fetal abnormalities in maternal cfDNA. The results demonstrated that T21 fetuses contain a perturbed epigenome and also indicated that fetal cfDNA might originate from fetal tissues other than placental chorionic villi. Robust covalent derivatization followed by targeted analysis of fetal DNA by sequencing or qPCR presents an attractive strategy that could help achieve superior sensitivity and specificity in prenatal diagnostics.
Subject: 1184 Genetics, developmental biology, physiology
3123 Gynaecology and paediatrics
NIPT
Fetal trisomy
Down syndrome
Cell-free DNA
DNA modification
5-Hydroxymethylcytosine
Covalent labeling
SIZE DISTRIBUTIONS
DIAGNOSIS
PLASMA
METHYLATION
ORDINATION
FRACTION
MARKERS
DOSAGE
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