This study examined the methylation difference in AIRE and RASSF1A between 
maternal and placental DNA, and the implication of this difference in the 
identification of free fetal DNA in maternal plasma and in prenatal diagnosis of 
trisomy 21. Maternal plasma samples were collected from 388 singleton 
pregnancies, and placental or chorionic villus tissues from 112 of them. 
Methylation-specific PCR (MSP) and methylation-sensitive restriction enzyme 
digestion followed by fluorescent quantitative PCR (MSRE + PCR) were employed to 
detect the maternal-fetal methylation difference in AIRE and RASSF1A. Diagnosis 
of trisomy 21 was established according to the ratio of fetal-specific AIRE to 
RASSF1A in maternal plasma. Both methods confirmed that AIRE and RASSF1A were 
hypomethylated in maternal blood cells but hypermethylated in placental or 
chorionic villus tissues. Moreover, the differential methylation for each locus 
could be seen during the whole pregnant period. The positive rates of fetal AIRE 
and RASSF1A in maternal plasma were found to be 78.1% and 82.1% by MSP and 94.8% 
and 96.9% by MSRE + PCR. MSRE + PCR was superior to MSP in the identification of 
fetal-specific hypermethylated sequences (P<0.05). Based on the data from 266 
euploidy pregnancies, the 95% reference interval of the fetal AIRE/RASSF1A ratio 
in maternal plasma was 0.33-1.77, which was taken as the reference value for 
determining the numbers of fetal chromosome 21 in 102 pregnancies. The accuracy 
rate in 98 euploidy pregnancies was 96.9% (95/98). Three of the four trisomy 21 
pregnancies were confirmed with this method. It was concluded that 
hypermethylated AIRE and RASSF1A may serve as fetal-specific markers for the 
identification of fetal DNA in maternal plasma and may be used for noninvasive 
prenatal diagnosis of trisomy 21.