A Study On The Establishment Of Non-invasive Prenatal Paternity Testing Methods Based On Differentially Methylated And Single Nucleotide Polymorphisms Markers

Background With the development of society, the needs of prenatal paternity testing gradually increased in recent years. However, conventional invasive sampling of amniotic fluid was restricted by gestation length, with the best time from 17 th week to 21 th week, and had a risk of resulting in fetal injury and miscarriage. Accordingly, it has been one of the hot areas of forensic genetics to find a safer and more flexible method for prenatal paternity testing. The discovery of cell- free fetal DN A(cffDNA) in maternal plasma opened up new possibilities for noninvasive prenatal paternity testing. C ffDN A can be detected starting from 6th week of gestion, and its content would increase during gestion, with an average about 10% of total plasma DN A and a clearance in 24 h or less after delivery. CffDNAs is ideal biological sample for non- invasive prenatal paternity testing, for it can be used in early pregnancy and not interfered by previous pregnancy. But directly testing for cffDNA in maternal plasma, backgrounded with a large number of maternal DNA, will be resulted in mixed genotypes, expect Y-specific sequences. Fortunately, epigenetics studies have found that the presence of differentially methylated regions(DMRs) between chorionic villi and maternal blood cells provides possibility for eliminating maternal DN A fom palsma. These methods firstly use MSRE(Methylation Sensitive Restriction Enzyme) to digest maternal DNA in plasma extensively, and then obtain fetal DN A with specific amplification of DMRs. However, genetic markers need to be typing for paternity testing, and the most suitable markers would be SNP(Single N ucleotide Polymorphism) since most cffDNA fragments existed with a length less than 150 bp. In addition, in order to achieve the demands of paternity testing, more SNP sites with relative low polymorphism will be typed. In a word, the combination of MSRE-PCR and SNaPshot to establish multiplex detection system has become a new strategy for non- invasive prenatal paternity testing.Objective The aim of the present study was to determine the flexibility of combining MSRE-PCR and SNaPshot to establish multiplex detection system for non- invasive prenatal paternity testing.Methods(1) According to some references and databases, we selected DMRs characterized by the following features: 1) Containing restriction sites combination HpaII and HinP1 I or Bst UI and HinP1 I. 2) All CpGs in MSRE recognition sequences methylated in fetal villi and unmethylated in maternal peripheral blood cells. 3) Containing Chinese Han population highly polymorphic SNP loci with minor allelic frequency no less than 0.2. 4) The maximum distance between SNP loci and the restriction sites is no more than 150 bp.(2) Primers for PCR amplification were designed uising the web-based software Primer 3. Then fetal villi DNA and corresponding maternal blood cells DNA were digested extensively with MSRE before PCR amplification. After separating amplification products using PAGE, MSRE specific DMRs were further selected by determining target bands existing in fetal villi DNA digested products instead of maternal blood cells DNA digested products.(3) The primers both for PC R amplification and the SBE reaction were designed with the web-based software Primer 3, and SBE primer 3? end base upstream or downstream adjacent to SNP loci were selected. The selected DMRs were separated into two groups by the type of MSRE. Specific primers were used to amplify target DNA fragments which contain SNP loci. After cleaned up with exonuclease I(Exo I) and shrimp alkaline phosphatase(SAP), the single base extension reactions were performed using the allelic-specific SBE primers. Then, 150 healthy unrelated individuals in Wuhan Han population were typed by using the established multiple detection system. In addition, 4 pregnant women' plasma DNA were also typed.Results(1) Fourteen MSRE-specific DMRs were selected which characterized by methylated in fetal villi and unmethylated in maternal blood cells.(2) The multiplex detection systems for typing 18 SNPs were successfully established, and all SNPs were polymorphic in Wuhan Han population.(3) The genotypes of all the four digested products from pregnant women plasma DNA were identical with the genotypes of corresponding fetal DNA.Conclusion The established multiplex detection system would be used to type the cffDNA in maternal plasma, and lay the groundwork for future research of non- invasive prenatal paternity testing.