Abstract
DNA methylation is an epigenetic marker that has been shown to vary significantly across different tissues. Taking advantage of the methylation differences between placenta-derived cell-free DNA and maternal blood, several groups employed different approaches for the discovery of fetal-specific biomarkers. The aim of this study was to analyse whole-genome fetal and maternal methylomes in order to identify and confirm the presence of differentially methylated regions (DMRs). We have initially utilized methylated DNA immunoprecipitation (MeDIP) and next-generation sequencing (NGS) to identify genome-wide DMRs between chorionic villus sampling (CVS) and female non-pregnant plasma (PL) and peripheral blood (WBF) samples. Next, using specific criteria, 331 fetal-specific DMRs were selected and confirmed in eight CVS, eight WBF and eight PL samples by combining MeDIP and in-solution targeted enrichment followed by NGS. Results showed higher enrichment in CVS samples as compared to both WBF and PL samples, confirming the distinct methylation levels between fetal and maternal DNA for the selected DMRs. We have successfully implemented a novel approach for the discovery and confirmation of a significant number of fetal-specific DMRs by combining for the first time MeDIP and in-solution targeted enrichment followed by NGS. The implementation of this double-enrichment approach is highly efficient and enables the detailed analysis of multiple DMRs by targeted NGS. Also, this is, to our knowledge, the first reported application of MeDIP on plasma samples, which leverages the implementation of our enrichment methodology in the detection of fetal abnormalities in maternal plasma.
Highlights
Chromosomal aneuploidies are the most common causes of genetic defects during the first trimester, which constitutes the major reason for pregnant women considering prenatal diagnosis (Hassold et al, 2007)
Despite efforts aimed at the discovery of fetalspecific markers that can be detected in all pregnancies irrespective of fetal gender and polymorphisms, the limited amount of cellfree fetal DNA (cffDNA) in the presence of a high maternal background presents a major challenge for the detection of fetal aneuploidies
Three wholeblood nonpregnant female samples (WBF), three chorionic villus sampling (CVS) and two PL samples were subjected to wholegenome MeDIPNGS analysis to enable genomewide identification of fetalspecific differentially methylated regions (DMRs)
Summary
Chromosomal aneuploidies are the most common causes of genetic defects during the first trimester, which constitutes the major reason for pregnant women considering prenatal diagnosis (Hassold et al, 2007). The discovery of cellfree fetal DNA (cffDNA) in maternal plasma was pivotal in the development of NIPT (Lo et al, 1997). Recent studies have shown that fetal DNA concentrations in the maternal circulation are estimated to be approximately 10% during the Downloaded from https://www.cambridge.org/core. Several research groups utilized the presence of cffDNA in maternal blood and were able to develop different approaches for the identification and utilization of fetalspecific biomarkers for NIPT. Despite efforts aimed at the discovery of fetalspecific markers that can be detected in all pregnancies irrespective of fetal gender and polymorphisms, the limited amount of cffDNA in the presence of a high maternal background presents a major challenge for the detection of fetal aneuploidies
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