Aneuploidy Detection in Mixed DNA Samples by Methylation-Sensitive Amplification and Microarray Analysis

Background: Cell-free fetal nucleic acid, believed to be derived from the placenta/trophoblast, is present in the plasma of pregnant women; however, its use for predictive genetic testing has been severely limited because the circulating fetal DNA is present in a small quantity and mixed with a much larger quantity of maternal DNA. Methods for detecting fetal aneuploidy from the cell-free fetal DNA in plasma are highly sought after, but proposed methods must take into account the small quantity and highly contaminated nature of the available fetal DNA. Methods: We developed a method for methylation-sensitive amplification of DNA suitable for use with small (approximately 1 ng) samples. We used this method in conjunction with 2-color microarray analysis with a custom-made array to investigate whether relative amplification, and hence relative methylation, could be evaluated for a large number of genomic loci. Results: Microarray assessment of genomic methylation accurately predicted the degree of methylation measured with bisulfite-conversion PCR and confirmed that DNA from first-trimester trophoblast was generally hypomethylated compared with whole-blood DNA. With a series of 3 samples in which 1 ng of DNA from a trisomic first trimester placenta was mixed with 9 ng of chromosomally normal peripheral blood DNA, we observed that the microarray signal associated with the trisomic chromosome was significantly different from that of the other chromosomes ( P < 0.001). Conclusions: This method has potential to be used for noninvasive detection of fetal aneuploidy.