Screening significantly hypermethylated genes in fetal tissues compared with maternal blood using a methylated-CpG island recovery assay-based microarray

Abstract

Background The noninvasive prenatal diagnosis procedures that are currently used to detect genetic diseases do not achieve desirable levels of sensitivity and specificity. Recently, fetal methylated DNA biomarkers in maternal peripheral blood have been explored for the noninvasive prenatal detection of genetic disorders. However, such efforts have covered only chromosomal aneuploidy, and fetal methylated DNA biomarkers in maternal whole blood for detecting single-gene diseases remain to be discovered. Methods To address this issue, we systematically screened significantly hypermethylated genes in fetal tissues and compared them with maternal peripheral blood potential in an attempt to detect fetal genes in maternal peripheral blood. First, the methylated-CpG island recovery assay combined with a CpG island array was performed for four fetus-toward placental tissues and the corresponding maternal peripheral bloods. Subsequently, direct bisulfite sequencing and combined bisulfite restriction analysis (COBRA) were carried out to validate the methylation status of the hypermethylated genes that were identified by the microarray analysis. Results Three hundred and ten significantly hypermethylated genes in the placental tissues were detected by microarray. From the top 15 hypermethylated genes detected by microarray, two were selected for sequencing validation in placental tissue and chorionic villus samples and four were selected for COBRA validation in four placental tissues, ten amniotic fluids and five chorionic villus samples. The six selected genes were confirmed to be hypermethylated in placental tissue and chorionic villus samples, but methylation of the genes could not be detected in the amniotic fluids. Conclusions Of the many hypermethylated genes and methylation sites that were found in the fetal tissues, some have great potential to be developed into molecular markers for noninvasive prenatal diagnosis of monogenic disorders. Further clinical studies are warranted to confirm these findings.

Epigenome‐wide DNA methylation assay reveals placental epigenetic markers for noninvasive fetal single‐nucleotide polymorphism genotyping in maternal plasma

“…Based on the 450K array analysis and independent validation by bisulfite genomic sequencing, priority was given for 27 CpG units located in 10 regions, with multiple differential methylated CpG sites overlapped with at least two MSRE recognizing sites and one polymorphic SNP, within a short stretch of DNA. Notably, three regions ( RASSF1A , SKI , and VAPA‐APCDD1 ) were previously described, thus emphasizing the robustness of our data. As a preliminary study, we selected three target regions involved in PSMB8 , SKI , and , detecting their presence in plasma samples before and after MRSE digestion.…”

“…Until now, a number of studies have reported placental epigenetic biomarkers on Chromosomes 13, 18, and 21, which are frequently found to be aneuploid in live births. However, only few investigations have focused on the candidate epigenetic targets on other chromosomes, neither of their potential applications besides fetal aneuploid diagnosis. Here, the quantitative Illumina BeadChip technique enables us to detect selected CpG sites in a genome‐wide basis at a single‐nucleotide resolution in virtually all genes, demonstrating the comprehensive differential methylation patterns in fetal placenta versus maternal blood cells.…”

Whole-genome fetal and maternal DNA methylation analysis using MeDIP-NGS for the identification of differentially methylated regions

“…The methylation status of the selected DMRs was also compared with previous studies that utilized methylation differences between fetal and maternal tissue for the identification of fetalspecific biomarkers. Common DMRs were found between our approach and DMRs identified using bisulphite conversion, methylationsensitive restriction digestion and microarrays (Old et al , 2007; Chim et al , 2008; Chu et al , 2009; Bunce et al , 2012; Yin et al , 2012; Lun et al , 2013; Hatt et al , 2015). More concordant results were observed between our study and DMRs identified using MeDIP approaches (Papageorgiou et al , 2009; Ioannides et al , 2014; Xiang et al , 2014).…”

Recurrent patterns of DNA methylation in theZNF154,CASP8, andVHLpromoters across a wide spectrum of human solid epithelial tumors and cancer cell lines

“…[13][14][15] Considerable effort has also been devoted to investigate mutational events that could explain cancer-related aberrant methylation through their effect on the epigenetic machinery. 16,17 Recently, the development of clinically relevant diagnostic tests for cancer based on methylation biomarkers measured in blood, urine, and other body fluids [18][19][20][21][22] have provided a glimpse into the biomedical applications that may become mainstream in the near future. 23 DNA methylation experiments performed by individual laboratories provide evidence for the identification of shared signatures of differential methylation that can separate tumors from normal controls.…”