Abstract
BackgroundNon-invasive prenatal testing of trisomy 21 (T21) is being actively investigated using fetal-specific epigenetic markers (EPs) that are present in maternal plasma. Recently, 12 EPs on chromosome 21 were identified based on tissue-specific epigenetic characteristics between placenta and blood, and demonstrated excellent clinical performance in the non-invasive detection of fetal T21. However, the disease-specific epigenetic characteristics of the EPs have not been established. Therefore, we validated the disease-specific epigenetic characteristics of these EPs for use in non-invasive detection of fetal T21.MethodsWe performed a high-resolution tiling array analysis of human chromosome 21 using a methyl-CpG binding domain-based protein (MBD) method with whole blood samples from non-pregnant normal women, whole blood samples from pregnant normal women, placenta samples of normal fetuses, and placenta samples of T21 fetuses. Tiling array results were validated by bisulfite direct sequencing and qPCR.ResultsAmong 12 EPs, only four EPs were confirmed to be hypermethylated in normal placenta and hypomethylated in blood. One of these four showed a severe discrepancy in the methylation patterns of T21 placenta samples, and another was located within a region of copy number variations. Thus, two EPs were confirmed to be potential fetal-specific markers based on their disease-specific epigenetic characteristics. The array results of these EPs were consisted with the results obtained by bisulfite direct sequencing and qPCR. Moreover, the two EPs were detected in maternal plasma.ConclusionsWe validated that two EPs have the potential to be fetal-specific EPs which is consistent with their disease-specific epigenetic characteristics. The findings of this study suggest that disease-specific epigenetic characteristics should be considered in the development of fetal-specific EPs for non-invasive prenatal testing of T21.
Highlights
Non-invasive prenatal testing of trisomy 21 (T21) is being actively investigated using fetal-specific epigenetic markers (EPs) that are present in maternal plasma
The aim of this study was to validate disease-specific epigenetic characteristics of EPs for the non-invasive prenatal testing (NIPT) of fetal T21 in whole blood from non-pregnant euploid women, whole blood from pregnant euploid women, euploid fetal placenta, and T21 fetal placenta using a high-resolution tiling array analysis of human chromosome 21 and to select the effective EPs that have the potential to be used as fetal-specific EPs for the NIPT of fetal T21
In this study, we validated that EP6 and EP7 have a hypermethylated pattern in placenta compared with blood, regardless of the presence or absence of disease and pregnancy and are detectable in maternal plasma
Summary
Non-invasive prenatal testing of trisomy 21 (T21) is being actively investigated using fetal-specific epigenetic markers (EPs) that are present in maternal plasma. 12 EPs on chromosome 21 were identified based on tissue-specific epigenetic characteristics between placenta and blood, and demonstrated excellent clinical performance in the non-invasive detection of fetal T21. We validated the disease-specific epigenetic characteristics of these EPs for use in non-invasive detection of fetal T21. The primary aim of prenatal testing is the diagnosis of fetal aneuploidies, such as trisomy 21 (T21, Down syndrome), trisomy 18. Current prenatal screening tests of T21 have greatly improved by using a combination of maternal serum markers and fetal sonographic markers such as nuchal translucency [3,4,5,6]. Positive screening results require confirmation with diagnostic testing, such as amniocentesis or chorionic villus sampling (CVS) [7]
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