An Exploration Study On Noninvasive Prenatal Paternity Test Based On Fetal Specific MRNA–SNP Markers

Backgroud As social openness continues to increase,there are more and more cases involving prenatal paternity testing.Currently,amniotic fluid is often used for testing,so there is a risk of miscarriage,fetal injury,etc.,but also limited by gestational age.Therefore,how to obtain fetal effective components in a non-invasive way has been a hot spot in forensic research.The discovery of cffDNA in pregnant women’s peripheral blood has really pointed the way for the exploration of noninvasive prenatal paternity testing(NIPPT).Compared with traditional invasive methods to obtain fetal components,fetal components obtained from the peripheral blood of pregnant women are safe,flexible,and basically eliminate the possibility of fetal injury.At present,due to the interference of a large number of maternal components,direct use of cffDNA in pregnant women’s peripheral blood to obtain fetal typing,most of which are maternal-fetal mixed genotypes,makes paternity testing difficult.Subsequent studies have found that there are cffmRNAs in the peripheral blood of pregnant women,providing another way to explore NIPPT.cffmRNA occurs early,and can be detected in the peripheral blood of the pregnant woman at the 4th week of pregnancy.It exists in the form of a submicron particle and is relatively stable.It is quickly cleared within 24 hours or less after delivery.Non-pregnancy male and female whole blood can not detect.Compared to cffDNA,cffmRNA is unique to the fetus and is not interfered by maternal components and is an ideal biomarker for NIPPT.Forensic parental testing requires genetic typing,commonly used genetic markers include STRs and SNPs.STR markers are basically located in the genomic non-coding region and therefore do not apply to cffmRNA typing markers.SNPs are the most abundant DNA sequence polymorphisms in the human genome.They are widely distributed,highly stable,and suitable for short segment typing.Therefore,SNP was selected as the cffmRNA typing marker.In summary,based on the maternal specificity of cffmRNA in maternal plasma,maternal peripheral plasma cffmRNA combined with SNP markers provides a new idea for us to explore NIPPT.Objective Exploring the Feasibility of Non-invasive Prenatal Fetal Parental Testing Using Fetal Specific mRNA Combined with SNP Markers in Pregnant Women’s Peripheral Plasma.Methods(1)Review the relevant literature to screen for placental-derived fetal-specific mRNA in peripheral blood plasma of pregnant women that can be used for noninvasive prenatal paternity testing.(2)Based on the candidate placenta-derived mRNA and chromosomal location selected from the literature,the NCBI genome browser was used to obtain the gene reference sequence corresponding to the candidate mRNA.(3)Entering the NCBI 1000 Genomes Browse,the Han population in southern China(CHS)minimum allele frequency≥0.15 as the screening criteria to obtain highly polymorphic SNP loci in the candidate mRNA region as candidates’mRNA-SNP marker.(4)According to the acquired mRNA gene sequence,Primer 3and AutoDimer software were used to design and test DNA-PCR,mRNA-PCR primers and single-base extension primers shared by DNA and mRNA,respectively.(5)The specificity of the placenta-derived mRNA were validated by two-step RT-PCR combined with SNaPshot technique.(6)According to the specific mRNA-SNP loci,DNA was used as a template to establish the SNaPshot detection system to investigate the gene frequency distribution in Hubei Han population and to detect the DNA-SNP genotypes of father,mother and amniotic fluid.(7)The peripheral blood samples of pregnant women during the second trimester were collected,TRIzol~?LS Reagent and RNeasy~?Mini Kit were used to extract and purify the plasma total RNA.Two-step RT-PCR combined with SNaPshot technology was used to detect the genotypes of target SNPs.The accuracy of the plasma SNP typing and the reliability of the detection system were determined by comparing the plasma SNP typing with the parental,maternal,and fetal typing.Results(1)The 9 candidate mRNAs(CRH,βhCG,PLAC4,KISS1,CD19,TFPI2,DHRS3,CSHL1 and KIR3DL2)are fetus-specific mRNAs derived from placenta.(2)Successfully constructed 9 mRNA-PCR composite amplification system and SNP composite detection system,9 SNPs(except KIR3DL2 rs652188)detected good genetic polymorphisms in Hubei Han population.(3)The maternal plasma cffmRNA-SNP genotype in the second trimester were completely consistent with the fetus.Conclusion Fetal specific mRNA combined with SNP markers in maternal plasma can be used for noninvasive prenatal paternity testing.