Non-invasive detection of fetal trisomy 21 using fetal epigenetic biomarkers with a high CpG density

Background: Non-invasive prenatal test of trisomy 21 (T21) is being researched using fetal specific epigenetic biomarkers present in maternal plasma. We applied a methyl-CpG binding domain-based protein (MBD) method based on epigenetic characteristics of fetal specific-methylated regions with a high CpG density in HLCS on chromosome 21 and RASSF1A on chromosome 3 for the non-invasive detection of fetal T21 and estimated the diagnostic accuracy of the method. Methods: A nested case-control study was conducted with maternal plasma collected from 50 pregnant women carrying 40 normal and 10 T21 fetuses. A MBD method was used for enrichment of methylated DNA regions in maternal plasma. The levels of methylated HLCS (M-HLCS) and methylated RASSF1A (M-RASSF1A) were simultaneously measured by multiplex qPCR. Results: Levels of M-HLCS and M-RASSF1A were obtained in all cases. Levels were not different according to fetal gender (p>0.05 in both). The level of M-HLCS was significantly increased in women with a T21 fetus compared with controls (p<0.001). The level of M-RASSF1A was not different between two groups (p>0.05). In non-invasive fetal T21 detection, the specificity of M-HLCS level and the epigenetic-epigenetic ratio (EER) using M-HLCS and M-RASSF1A levels were 82.5% and 92.5%, respectively, at 90.0% sensitivity. Conclusions: Our findings suggest that the EER may be useful as a potential biomarker for the non-invasive detection of fetal T21, regardless of fetal gender. The MBD method can be used as an effective tool in the detection of methylated fetal specific markers with a high CpG density in maternal plasma.