Fetal-specific DNA methylation ratio permits noninvasive prenatal diagnosis of trisomy 21

Abstract

The trials performed worldwide toward noninvasive prenatal diagnosis (NIPD) of Down's syndrome (or trisomy 21) have shown the commercial and medical potential of NIPD compared to the currently used invasive prenatal diagnostic procedures. Extensive investigation of methylation differences between the mother and the fetus has led to the identification of differentially methylated regions (DMRs). In this study, we present a strategy using the methylated DNA immunoprecipitation (MeDiP) methodology in combination with real-time quantitative PCR (qPCR) to achieve fetal chromosome dosage assessment, which can be performed noninvasively through the analysis of fetal-specific DMRs. We achieved noninvasive prenatal detection of trisomy 21 by determining the methylation ratio of normal and trisomy 21 cases for each tested fetal-specific DMR present in maternal peripheral blood, followed by further statistical analysis. The application of this fetal-specific methylation ratio approach provided correct diagnosis of 14 trisomy 21 and 26 normal cases.

Figure 1. Schematic illustration of the fetal-specific DNA methylation ratio approach.

The fetus with trisomy 21 has an extra copy of the fetal-specific methylated region compared to the normal fetus. DNA methylation enrichment followed by real-time qPCR of a fetal-specific methylated region, can lead to relative quantification of the amount of fetal DNA in normal and trisomy 21 cases.

Figure 2. DNA methylation ratio values obtained from the 12 DMRs.

The y axis represents the methylation ratio value. Each color dot represents the ratio value obtained from one of the 12 DMRs tested (EP1 to EP12). (a) DNA methylation ratio values obtained from the normal cases P6 and P13. (b) DNA methylation ratio values obtained from the trisomy 21 cases P29 and P35.

Figure 3. BoxPlot representation of the results obtained from four DMRs.

EP1, EP4, EP7 and EP10 in 20 normal and 20 trisomy 21 cases. The boxplots depict the 5-number summaries, namely the minimum and maximum values, the upper (Q3) and lower (Q₁) quartiles, and the median. The median is identified by a line inside the box. The length of the box represents the interquartile range (IQR = Q₃ – Q₁). Values more than three IQR’s from either end of the box are labeled as extremes and denoted by an asterisk (*).Values more than 1.5 IQR’s but less than 3 IQR’s from either end of the box are labeled as outliers (o).