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

Clinical Epigenetics

Kaarel Krjutškov¹  Andres Salumets²  Priit Paluoja³  Milda Narmontė⁴  Juozas Gordevičius⁴  Povilas Gibas⁴  Vita Tomkutė⁴  Kotryna Kvederavičiūtė⁴  Edita Kriukienė⁵ 

[1] Competence Centre On Health Technologies, Teaduspargi 13, 50411, Tartu, Estonia;Department of Obstetrics and Gynaecology, Institute of Clinical Medicine, University of Tartu, L. Puusepa 8, 50406, Tartu, Estonia;Competence Centre On Health Technologies, Teaduspargi 13, 50411, Tartu, Estonia;Department of Obstetrics and Gynaecology, Institute of Clinical Medicine, University of Tartu, L. Puusepa 8, 50406, Tartu, Estonia;Department of Obstetrics and Gynecology, University of Helsinki and Helsinki University Hospital, HUS, PO Box 140, 00029, Helsinki, Finland;Estonian Genome Center, Institute of Genomics, University of Tartu, Riia 23b, 51010, Tartu, Estonia;Competence Centre On Health Technologies, Teaduspargi 13, 50411, Tartu, Estonia;Faculty of Medicine, University of Helsinki, 00014, Helsinki, Finland;Department of Biological DNA Modification, Institute of Biotechnology, Vilnius University, Saulėtekio av. 7, 10257, Vilnius, Lithuania;Department of Biological DNA Modification, Institute of Biotechnology, Vilnius University, Saulėtekio av. 7, 10257, Vilnius, Lithuania;Institute of Biotechnology, Vilnius University, Saulėtekio av. 7, 10257, Vilnius, Lithuania;

关键词: NIPT;    Fetal trisomy;    Down syndrome;    Cell-free DNA;    DNA modification;    5-Hydroxymethylcytosine;    Covalent labeling;   

DOI  :  10.1186/s13148-020-00938-x

来源: Springer

【 摘 要 】

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.

【 授权许可】

CC BY