The chromosome 21 marker was hypermethylated HLCS, and the reference markers on chromosome 3 and chromosome Y were hypermethylated RASSF1A and male-specific ZFY, respectively.
Two pairs of placental tissue and maternal blood cell samples were used to check the performance of the HLCS and RASSF1A assays with and without enzyme digestion.
Results from the BstUI-digested samples of placental DNA showed that about 60%-70% of the HLCS and RASSF1A molecules remained detectable, compared with the mock-digested samples.
Analysis of postdelivery maternal plasma revealed extremely low levels of both the HLCS and RASSF1A molecules after BstUI digestion (Table S2B in the online Data Supplement).
The HLCS and RASSF1A assays were applied to 10 euploid and 12 T21 placental DNA samples from pregnancies with male fetuses.
The same restriction enzyme-digested placental DNA samples used for the HLCS and RASSF1A comparison were subjected to the ZFY digital PCR analysis (Fig.
After BstUI digestion, each DNA sample was analyzed with the HLCS and ZFY assays in at least 6 panels on the microfluidics chips (Fig.
In contrast, all 5 maternal plasma samples from T21 pregnancies had a ratio of HLCS to ZFY that was greater than the upper reference limit (P < 0.
In the first part of this study, we successfully identified a new hypermethylated fetal DNA marker on chromosome 21, the promoter region of the HLCS gene.
We then used HLCS to demonstrate that the novel EGG approach is a feasible method for analyzing fetal chromosome dosage.
the ratio of hypermethylated HLCS to hypermethylated RASSF1A.
In fact, because both HLCS and ZFY are fetal-specific markers in maternal plasma, it should be possible to detect fetal aneuploidy in maternal plasma by EGG analysis with nondigital platforms, such as conventional real-time quantitative PCR.