Screening And Identification Of Differentially Methylated Markers Between Maternal And Fetal DNA

Research background Prenatal paternity testing plays an important role in forensic paternity testing. Traditional prenatal paternity testing usually requires puncture extraction of the amniotic fluid, umbilical cord blood or chorionic villus sampling, but these invasive sampling exist the risk of abortion, intrauterine infection and fetal injury, and may be limited by pregnancy time. For a long time, to explore the noninvasive prenatal paternity test method has been an important research topic in the field of forensic genetics. Studies have found that maternal blood contains a variety of fetal cells and fetal free nucleic acids. The former content in maternal blood is too little, and with the difficulty of separation and enrichment, which resulting in a prenatal paternity test application of limited value. The latter brings a new opportunity for non invasive fetal paternity testing in forensic science. At present, although the use of maternal blood in the cffDNA for fetal paternity has made some progress, but there are still the following problems:(1) CffDNA was so less and it is difficult to enrich. In addition, it is coupled with a small fragment, which resulting in the limited detection of fetal genetic markers; (2) Due to interference of maternal DNA, test results were mixed genotype of the mother and fetus, which making the evaluation of the patriarchal judgment become more complex. The latest research results of the apparent genetics have confirmed that there are many DMR between the maternal and fetal DNA. The discovery of these DMR can not only lay the foundation for non-invasive prenatal diagnosis of fetal, but also provide a new technical means for forensic noninvasive fetal paternity testing. Different from the clinical noninvasive prenatal diagnosis, forensic paternity testing required genotyping. Thus, the mother SNP loci in DMR naturally become the object of concern. Combined analysis of differentially methylated markers and SNP sites become a new strategy for noninvasive prenatal paternity testing. Based on this, screening and identification of differential DNA methylation markers between mother and fetus will become a very important basic work to conduct noninvasive prenatal paternity testing using epigenetic markers.Objective To screen the DMR with significant differences between maternal fetal DNA methylation and with better frequency of SNP sites in genome-wide to provide candidate marker for noninvasive fetal prenatal paternity testing by using epigenetics.Methods Maternal blood cells (MBC) and fetal placental chorionic (CVS) were as the research object to systematically detect the difference of methylation level of maternal DNA by high throughput NimbleGen HG18 CpG Promoter methylation chip. The results of the chip were verified by the method of the pyrosequencing. A number of SNP markers and methylation sensitive restriction sites of the enzyme in accordance with the amplification system were selected. Finally, further validation was made in maternal plasma to evaluate the specificity, stability and sensitivity of the fetal epigenetic markers in maternal circulation.Results (1) A total of 185 highly methylated regions and 303 methylated regions were found in PeakDMvalue>0.5. In these 488 differentially methylated fragments,75 (15.37%) were located in the CpG island region and 413 (84.63%) were located in the promoter region. The promoter region occupies most of the differentially methylated fragments.Differentially methylated fragments were randomly distributed on all chromosomes. Chromosome 1 contains the most differentially methylated regions:42. Next is the chromosome 19:35, chromosome 5:30, chromosome 22:30, chromosome 17:27, chromosome 11:25, chromosome 7:24. Database was used to search for SNPs in the screening of 488 maternal DMR, and the population's gene frequency distribution was investigated to search for SNP sites with high polymorphism and non linkage in maternal DMR.488 maternal DMR were screened one by one in NCBI and SPS database by us. The number of SNP sites that DM value>0.5 and SNP frequency>0.2 was 91. (2) We considered the DM values, differences in maternal and fetal fragments, SNP frequencies and enzyme cutting site positions, and eventually 15 fragments were selected to validation analysis of the subsequent pyrosequencing. In these 15 fragments, there were 3 fragments that consistent with the microarray results andcontaining the enzyme sites close to the SNP site. (3) For the chr10:101281722-101282479 (NCBI36/hg18) piece of six samples, the maternal fragments were completely cut by BstUI and HinP1I while the fragments of fetal villi are not cut, clear difference between them can make effective distinction. The other two pieces chr9:138021600-138022048 (NCBI36/hg18) and chr17:34251133-34251505 (NCBI36/hg18) of enzyme situation reflects a certain number of individual differences.Conclusion This subject has found and validated 1 methylation fragments with maternal differences in the whole genome wide range by methylation microarray screening. This fragment can be used in prenatal paternity testing as a specific marker for distinguishing between the fetus and the mother. Other two DMR have a certain number of individual differences only meet part of prenatal paternity testing requirements.