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

BACKGROUND: Massively 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 feta...

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Autores principales: Gordevičius, Juozas, Narmontė, Milda, Gibas, Povilas, Kvederavičiūtė, Kotryna, Tomkutė, Vita, Paluoja, Priit, Krjutškov, Kaarel, Salumets, Andres, Kriukienė, Edita
Formato: Online Artículo Texto
Lenguaje:English
Publicado: BioMed Central 2020
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7574562/
https://www.ncbi.nlm.nih.gov/pubmed/33081811
http://dx.doi.org/10.1186/s13148-020-00938-x
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author Gordevičius, Juozas
Narmontė, Milda
Gibas, Povilas
Kvederavičiūtė, Kotryna
Tomkutė, Vita
Paluoja, Priit
Krjutškov, Kaarel
Salumets, Andres
Kriukienė, Edita
author_facet Gordevičius, Juozas
Narmontė, Milda
Gibas, Povilas
Kvederavičiūtė, Kotryna
Tomkutė, Vita
Paluoja, Priit
Krjutškov, Kaarel
Salumets, Andres
Kriukienė, Edita
author_sort Gordevičius, Juozas
collection PubMed
description BACKGROUND: Massively 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. METHODS: We 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. RESULTS: We 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. CONCLUSIONS: uTOP-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.
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spelling pubmed-75745622020-10-21 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 Clin Epigenetics Research BACKGROUND: Massively 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. METHODS: We 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. RESULTS: We 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. CONCLUSIONS: uTOP-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. BioMed Central 2020-10-20 /pmc/articles/PMC7574562/ /pubmed/33081811 http://dx.doi.org/10.1186/s13148-020-00938-x Text en © The Author(s) 2020 Open AccessThis article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article's Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article's Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated in a credit line to the data.
spellingShingle Research
Gordevičius, Juozas
Narmontė, Milda
Gibas, Povilas
Kvederavičiūtė, Kotryna
Tomkutė, Vita
Paluoja, Priit
Krjutškov, Kaarel
Salumets, Andres
Kriukienė, Edita
Identification of fetal unmodified and 5-hydroxymethylated CG sites in maternal cell-free DNA for non-invasive prenatal testing
title Identification of fetal unmodified and 5-hydroxymethylated CG sites in maternal cell-free DNA for non-invasive prenatal testing
title_full Identification of fetal unmodified and 5-hydroxymethylated CG sites in maternal cell-free DNA for non-invasive prenatal testing
title_fullStr Identification of fetal unmodified and 5-hydroxymethylated CG sites in maternal cell-free DNA for non-invasive prenatal testing
title_full_unstemmed Identification of fetal unmodified and 5-hydroxymethylated CG sites in maternal cell-free DNA for non-invasive prenatal testing
title_short Identification of fetal unmodified and 5-hydroxymethylated CG sites in maternal cell-free DNA for non-invasive prenatal testing
title_sort identification of fetal unmodified and 5-hydroxymethylated cg sites in maternal cell-free dna for non-invasive prenatal testing
topic Research
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7574562/
https://www.ncbi.nlm.nih.gov/pubmed/33081811
http://dx.doi.org/10.1186/s13148-020-00938-x
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