MeDIP combined with in-solution targeted enrichment followed by NGS: Inter-individual methylation variability of fetal-specific biomarkers and their implementation in a proof of concept study for NIPT

PLoS One. 2018 Jun 11;13(6):e0199010. doi: 10.1371/journal.pone.0199010. eCollection 2018.


DNA methylation is the most characterized epigenetic process exhibiting stochastic variation across different tissues and individuals. In non-invasive prenatal testing (NIPT) fetal specific methylated regions can potentially be used as biomarkers for the accurate detection of fetal aneuploidies. The aim of this study was the investigation of inter-individual methylation variability of previously reported fetal-specific markers and their implementation towards the development of a novel NIPT assay for the detection of trisomies 13, 18, and 21. Methylated DNA Immunoprecipitation (MeDIP) combined with in-solution targeted enrichment followed by NGS was performed in 29 CVS and 27 female plasma samples to assess inter-individual methylation variability of 331 fetal-specific differentially methylated regions (DMRs). The same approach was implemented for the NIPT of trisomies 13, 18 and 21 using spiked-in (n = 6) and pregnancy samples (n = 44), including one trisomy 13, one trisomy 18 and four trisomy 21. Despite the variability of DMRs, CVS samples showed statistically significant hypermethylation (p<2e-16) compared to plasma samples. Importantly, our assay correctly classified all euploid and aneuploid cases without any false positive results (n = 44). This work provides the starting point for the development of a NIPT assay based on a robust set of fetal specific biomarkers for the detection of fetal aneuploidies. Furthermore, the assay's targeted nature significantly reduces the analysis cost per sample while providing high read depth at regions of interest increasing significantly its accuracy.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Aneuploidy
  • Biomarkers / analysis*
  • Chromosomes, Human, Pair 18
  • Chromosomes, Human, Pair 21
  • DNA / chemistry
  • DNA / isolation & purification
  • DNA / metabolism*
  • DNA Methylation
  • Down Syndrome / genetics
  • Female
  • Fetus / metabolism
  • High-Throughput Nucleotide Sequencing
  • Humans
  • Immunoprecipitation
  • Pregnancy
  • Prenatal Care*
  • Sequence Analysis, DNA


  • Biomarkers
  • DNA

Grant support

This work has received funding from the European Research Council (ERC) under 7th Framework Programme under grant agreement No 322953 for the project: A Novel Non-Invasive Prenatal Diagnosis of Genetic Disorders. Furthermore, the authors, Marios Ioannides, Charalambos Loizides, Kyriakos Tsangaras, Achilleas Achilleos, Petros Mina, Elena Kypri, Skevi Kyriacou, George Koumbaris and Philippos C Patsalis are employed by NIPD Genetics Ltd. NIPD Genetics Ltd., provided support in the form of salaries for authors MI, CL, KT, AA, PM, EK, SK, GK and PCP. Its role was mainly in the design of the study, data analysis, troubleshooting, editing of the manuscript and project supervision. The specific roles of these authors are articulated in the ‘author contributions’ section.