Fig 1.
Overview of the Illumina 450K beadchip data filtering process.
Methylation data (β-values) for the 485,777 CpGs were filtered in five steps. First, probes for which no β-values were obtained for one or more samples were removed, resulting in a complete dataset for 471,956 CpGs. Second, 144,273 differentially methylated CpGs (DMCs) between maternal Blood Cells (MBC) and Chorionic villus samples (CVS) were identified using a False Discovery Rate (FDR) of <0.05 and delta β>0.2.Third, a more stringent definition of DMCs where MBC samples are hypomethylated (mean β<0.25) and CVS samples are hypermethylated (mean β>0.75), or vice versa, dramatically reduced the number of DMCs to 5,956. Fourth, requiring DMCs to overlap a restriction site for a methylation–sensitive restriction enzyme (MSRE), further reduced the number of DMCs to 2,184. Fifth, only DMCs where at least 10 sampleswithin the group pass the hypo- and hyper-methylation thresholds are included, resulting in 958 MSRE-overlapping CpGs with markedly different and highly consistent methylation status between MBC- and CVS-samples.
Fig 2.
Ideogram of the 958 differentially methylated CpGs (DMCs) between Maternal blood cells (MBC) and Chorionic villus samples (CVS).
DMCs that are hypermethylated in MBCs and hypomethylated in CVS are shown in red., whereas DMCs hypomethylated in MBC and hypermethylated in CVS are blue. Chromosomes 13, 18 and 21 have been marked with a black triangle. DMCs on these chromosomes holds the possible use for diagnosis of the three main trisomies; Trisomy 13, 18 and 21.
Fig 3.
Heatmap visualization of the DNA methylation of 44 differentially methylated CpGs (DMCs) on Chromosome 13, 18 and 21.
Rows represent CpG targets, columns represent samples. Each cell is colored according to the level of methylation (β-value).Blue; low β-values, yellow; intermediate β-values, red; high β-values.
Table 1.
Identified candidate biomarkers on chromosome 13, 18 and 21.
The table lists for each CpG target the CpG target identification number (illumina), chromosome, nearest gene name, mean methylation levels(β-values) for Maternal Blood Cells(MBC) and Chorionic Villus Samples(CVS), the difference (delta β) between MBC and CVS, and the restriction site for a methylation-sensitive restriction enzyme(MSRE).
Table 2.
Identified epigenetic biomarkers located in micro-deletion-syndromes.
Differentially methylated CpG sites(DMCs))between Maternal Blood Cells(MBCs) and Chorionic Villi samples(CVS) located in DNA regions underlying micro-deletion-syndromes. The table lists chromosome, start, end and region size for the deletion syndromes, and numbers of identified DMCs in each region.
Fig 4.
Scatterplot displaying data reproducibility.
The beadchip encompass 65 highly polymorphic single nucleotide polymorphisms (SNPs) of the β-vaules for 65 polymorphic SNPs for replicate samples. 4 samples were analysed in duplicates. Two MBC- and two CVS-samples underwent independent bisulphite conversion and were analysed on different bead chip arrays. Spearman correlation coefficient (ρ) for each pair of replicates is shown. Diagonal red line represents complete correlation (ρ = 1.00). Methylation levels (β-value) of 0,0, 0.5, and 1.0 shows clear distinct patterns of homozygous or heterozygous methylation.