Sites of differential DNA methylation between placenta and peripheral blood: molecular markers for noninvasive prenatal diagnosis of aneuploidies

Abstract

The use of epigenetic differences between maternal whole blood and fetal (placental) DNA is one of the main areas of interest for the development of noninvasive prenatal diagnosis of aneuploidies. However, the lack of detailed chromosome-wide identification of differentially methylated sites has limited the application of this approach. In this study, we describe an analysis of chromosome-wide methylation status using methylation DNA immunoprecipitation coupled with high-resolution tiling oligonucleotide array analysis specific for chromosomes 21, 18, 13, X, and Y using female whole blood and placental DNA. We identified more than 2000 regions of differential methylation between female whole blood and placental DNA on each of the chromosomes tested. A subset of the differentially methylated regions identified was validated by real-time quantitative polymerase chain reaction. Additionally, correlation of these regions with CpG islands, genes, and promoter regions was investigated. Between 56 to 83% of the regions were located within nongenic regions whereas only 1 to 11% of the regions overlapped with CpG islands; of these, up to 65% were found in promoter regions. In summary, we identified a large number of previously unreported fetal epigenetic molecular markers that have the potential to be developed into targets for noninvasive prenatal diagnosis of trisomy 21 and other common aneuploidies. In addition, we demonstrated the effectiveness of the methylation DNA immunoprecipitation approach in the enrichment of hypermethylated fetal DNA.

Figure 1

DNA methylation enrichment of CHR21(A) region assayed by oligonucleotide array. Methylation difference between third trimester placenta and whole blood and individual methylation status of whole blood, first trimester and third trimester placental DNA samples.

Figure 2

Comparison of the DNA methylation enrichment of CHR21(A) from oligonucleotide arrays and real-time quantitative PCR using whole blood, first trimester and third trimester placental DNA samples. The y axis indicates the relative fold enrichment of placenta when compared with whole blood DNA sample and the x axis indicates the chromosomal position in bp. The gray lines represent the oligonucleotides covering the specific region on chromosome 21 whereas the black lines represent the PCR products [CHR21(A1) and CHR21(A2)] Biomarkers, Genomics, Proteomics, and Gene Regulation when real-time quantitative PCR was applied. The dotted lines represent the results obtained from a first trimester placenta whereas the solid lines represent the results obtained from a third trimester placenta.

Figure 3

DNA methylation enrichment of CHR21(B4) by real-time quantitative PCR on MeDiP and input replicated DNA samples. Open bars, whole blood samples; gray bars, first trimester placental DNA samples; solid black bars, third trimester placental DNA samples. The error bars indicate the SD between technical replicates. WB, whole blood; PL, placenta.

Figure 4

DNA methylation enrichment of CHR13(HYP1) using real-time quantitative PCR. Open bars, input DNA compared with whole blood; solid black bars, immunoprecipitated DNA compared with whole blood; 1PL and 2PL, third trimester placentas; 3PL, first trimester placenta; WB, whole blood; PL, placenta.