This report describes the first identification and characterization of three 
chromosome-21-specific DNA sequences (and reference sequences from other 
chromosomes) that are differentially methylated between peripheral blood and 
placental tissue, with the aim of providing epigenetic biomarkers for 
quantifying cell-free fetal DNA in maternal plasma. To select sequences to be 
screened for differential methylation, three strategies were adopted: (i) 
investigating promoters of highly differentially expressed genes; (ii) choosing 
'random' promoter regions; and (iii) choosing 'random' non-promoter regions. 
Over 200 pre-selected DNA sequences were screened using a methylation-specific 
restriction enzyme assay. Differentially methylated sequences located at 21q22.3 
(AIRE, SIM2 and ERG genes), 1q32.1 (CD48 gene and FAIM3 gene), 2p14 (ARHGAP25 
gene) and 12q24 (SELPLG gene) were identified. Bisulphite conversion confirmed 
that CpG sites within the AIRE promoter region are highly differentially 
methylated, and optimized methylation-specific primers for this region that are 
highly specific for placental DNA were devised. Next, it was shown that the 
methylation status of chorionic villus sample DNA from first trimester 
pregnancies matched the hypermethylated state of term placenta. Thus there is no 
indication of a difference in methylation status between early and term 
pregnancy for the sequences tested. The identified sequences constitute 
candidate biomarkers for non-invasive prenatal diagnosis of Down syndrome.