Noninvasive Prenatal Screen Of Trisomy-21 Using Maternal Plasma Fetal Free MRNA Allelic Ratio

Trisomy 21 is one of the most frequent chromosome disorder, and mainly performed as low intelligence, multiple organ dysfunction.The prenatal diagnosis of Trisomy 21 has been the key research content of obstetric medical workers.Karyotyping of fetal cells is the gold standard for prenatal diagnosis of Trisomy21.The procedures to obtain fetal cells such as chorionic villus sampling and amniocentesis are invasive, and may impose potential risks to the mother or the fetus.Recently, the discovery of cell-free fetal nucleic acids in maternal circulation opens a new window for noninvasive prenatal diagnosis, which has been moved into the application of testing fetal genetic disorders. The presence of fetal m RNA in maternal plasma was first demonstrated by Poon et al. which was the basis of non-invasive prenatal diagnosis of Trisomy 21. MLPA is a new quantitative method that was used for noninvasive prenatal diagnostic of Trisomy 21,which makes use of the genetic single-nucleotide polymorphism(SNP) of cell-free fetal m RNA in maternal peripheral blood. The purpose of this research was sing DNA sequencing and PCR-RFLP to determine the status of 10 SNPs of fetal specific genes PLAC4 and COL6A2 in Han population samples collected in He Nan Provice of China,and then calculated their heterozygote frequencies; and detected the m RNA expression of PLAC4 and COL6A2 to determine the optimal gastational age; and selected 5 SNPswith high heterozygosity to diagnosis 40 blood samples by using RT-MLPA.ObjectiveThrough the analysis of this research area in henan province han crowd PLAC4 and COL6A2 genes on chromosome 21 characteristics of multiple single nucleotide polymorphisms, and cell-free fetal m RNA expression level in different gestational age of maternal peripheral blood,Explore MLPA for 21- the region fetal non-invasive prenatal screening value of three body syndrome.Materials and Methods1 Materials①The first part: Slected 500 patients consisted of 250 men and 250 women undergoing investigation at The Third Affiliated Hospital of Zhengzhou University were recruited with informed consent between 2013.6and 2013.12 randomly. About5 m L of peripheral blood was collected and all patients recruited for this study were born in He Nan Provice of China. ②The second part: Randomly cllected 30 Maternal peripheral blood samples of pregnant women who visited the Department of Obstetrics and Gynecology of The Third Affiliated Hospital of Zhengzhou University at 8、10、12、14、16 weeks of gastation with informed consent. ③The third part: We selected 10 pregnancies with a euploid fetus and 10 pregnancies with a trisomy-21 fetus and cllected 5ml of maternal peripheral blood into EDTA-containing tubes. This study was approved by the Medical Ethics Committees of The Third Affiliated Hospital of Zhengzhou University.2 Methods① The first part: Using DNA sequencing and polymerase chain reactionrestriction fragment length polymorphism(PCR-RFLP),we determined ten the status of m RNA-SNPs of fetal specific genes PLAC4 and COL6A2 in 500 Han population samples collected in He Nan Provice of China, and then calculated their heterozygotefrequencies; ②The second part: Maternal peripheral blood samples of pregnant women at 8、10、12、14、16 weeks of gastation were collected and detected the m RNA expression of PLAC4 and COL6A2 by Real-time fluorescent quantitative reverse transcription polymerase chain reaction(RT-PCR);③The third part: We selected 20 pregnancies with a euploid fetus and 20 pregnancies with a trisomy-21 fetus to perform noninvasive prenatal diagnosis of Trisomy-21 by Real-time multiplex ligation-dependent probe amplification(RT-MLPA).Results1 The first part.The heterozygosity of every SNP from 500 persons in Henan province was:1)PLAC4: rs59066201(61.6%), rs8130833(39.2%), rs4818219(34.8%),rs9977003(21.6%), rs7844(19.2%).2)COL6A2: rs7717(49.6%), rs1044598(53.4%), rs559(50.2%), rs3088026(12.8%), rs1042917(45.6%):3) The heterozygosity of rs59066201 was no reported in NCBI; Compared to NCBI Hap Map- HCB, the heterozygosity of rs8130833, rs9977003, rs7844 were significantly different(P < 0.05), and the other SNPs were no difference(P > 0.05).2 The second part Peripheral blood of 30 pregnant women in 8 gastation weeks, 10 weeks, 12 weeks, 14 weeks, 16 weeks were collected,and the expression level of them showed an increasing trend with gestational age;1) The expression level of PLAC4 m RNA was 7.22±1.05 、 8.02±1.41 、9.51±1.69、11.33±2.11、13.31±2.582) The expression level of COL6A2 m RNA was 8.95±1.28 、 11.19±1.36 、15.0±1.58、16.87±1.72、18.96±2.79;3 The third part Five high heterozygosity SNPs were selected while the population coverage was 97.6%{[1-(1-49.6%)×(1-53.4%)×(1-50.2%)×(1-61.6%)×(1-45.6%)]=97.6%},to screen 20 Trisomy-21 fetuses confirmed by karyotyping of fetal cells obtained by amniocentesis and 20 normal fetuses with sensitivity of 87.5%and specifity of 100%.Conclusions1 The 10 SNPs: rs7717, rs559, rs1044598, rs59066201, rs1042917 in our study were high heterozygosity and the population coverage was 97.6%, and the 5 SNPs can be used non-invasive prenatal screening;2 PLAC4 and COL6A2 m RNA were indeed detected in maternal plasma, The RNA-SNP allelic ratio can promisingly be used for noninvasive prenantal Screen of Trisomy-21 between 8 to 16 weeks of pregnant women, when fetal specific SNPs are heterozygous.