Differential DNA Methylation as a Tool for Noninvasive Prenatal Diagnosis (NIPD) of X Chromosome Aneuploidies

Abstract

The demographic tendency in industrial countries to delay childbearing , coupled with the maternal age effect in common chromosomal aneuploidies and the risk to the fetus of invasive prenatal diagnosis, are potent drivers for the development of strategies for noninvasive prenatal diagnosis. One breakthrough has been the discovery of differentially methylated cell-free fetal DNA in the maternal circulation. We describe novel bisulfite conversion-and methylationsensitive enzyme digestion DNA methylation-related approaches that we used to diagnose Turner syndrome from first trimester samples. We used an Xlinked marker , EF3 , and an autosomal marker, RASSF1A , to discriminate between placental and maternal blood cell DNA using real-time methylationspecific PCR after bisulfite conversion and real-time PCR after methylation-sensitive restriction digestion. By normalizing EF3 amplifications versus RASSF1A outputs , we were able to calculate sex chromosome/ autosome ratios in chorionic villus samples , thus permitting us to correctly diagnose Turner syndrome. The identification of this new marker coupled with the strategy outlined here may be instrumental in the development of an efficient , noninvasive method of diagnosis of sex chromosome aneuploidies in plasma

Discovery of epigenetic biomarkers for the noninvasive diagnosis of fetal disease

“…Previous studies have identified a number of potentially significant DMRs in the context of noninvasive fetal diagnosis. 23,25,26,28,30,[37][38][39] The study presented here is the first to describe the identification of DMRs between CVS and MBC in a genome-wide fashion and, to our knowledge, is the first to confirm CpG-specific differential DNA methylation using targeted single locus assays directed towards chromosome 13 markers using samples from multiple pregnancies.…”

“…As demonstrated previously, CpG sites identified in this way are potential biomarkers for the selective amplification of fetal DNA from maternal plasma and subsequent detection of fetal trisomy. 23,28,29,31,35 When analyzing our data, we focused our efforts on identifying differentially methylated CpG sites located on chromsomes 13, 18, 21, X, and Y because these are most commonly found aneuploidies in human populations. Therefore, biomarkers on these chromosomes have the greatest potential clinical utility.…”

“…[22][23][24][25][26][27] These unique features of placental nucleic acids can then be used for the selective recovery of trophoblast-derived nucleic acid and subsequent fetal genetic analysis. Selective recovery may be achieved, for example, via methylation-specific PCR following bisulphate conversion 23,28,29 or methylation-sensitive restriction digestion 19,28,30 or via immunoprecipitation of methylated fetal DNA. 25,31 Aneuploidy can then be confirmed or excluded by quantifying levels of locus-specific markers on chromosomes of interest, for example, via the analysis of allelic ratios at heterozygous fetal polymorphisms, 23 locus-specific quantitative PCR, 19,28,29,31 or via microarray-based analysis.…”

“…Selective recovery may be achieved, for example, via methylation-specific PCR following bisulphate conversion 23,28,29 or methylation-sensitive restriction digestion 19,28,30 or via immunoprecipitation of methylated fetal DNA. 25,31 Aneuploidy can then be confirmed or excluded by quantifying levels of locus-specific markers on chromosomes of interest, for example, via the analysis of allelic ratios at heterozygous fetal polymorphisms, 23 locus-specific quantitative PCR, 19,28,29,31 or via microarray-based analysis. 30 In the current study, we have utilized a commercially available microarray-based platform to identify cytosineguanine dinucleotides (CpGs) that are differentially methylated between maternal blood cells (MBCs) and chorionic villus tissue.…”

High-Throughput Massively Parallel Sequencing for Fetal Aneuploidy Detection from Maternal Plasma

“…The greatest advantage of a whole genome approach is that it enables the impartial analysis of the entire genome, enabling detection of genomic aberrations without a priori region selection. Region-specific methods theoretically support higher throughput while still achieving acceptable performance for the two most common trisomies (trisomy 21 and trisomy 18); however, these targeted assays, including those not based on Massively Parallel Sequencing (MPS) [10]–[12], are restricted by the significant amount of re-development required when additional content, for example less frequent trisomies or sex chromosome aneuploidies, is introduced. Additionally, it remains to be seen how well these targeted methods can identify events such as partial trisomies or other large copy number variations.…”