Research On The Application Of Quantitative Pyrosequencing For Rapid Diagnosis Of Down's Syndrome

DOWN'S syndrome is one of the most common chromosome aneuploidies for fetal. The main charachater is severe congenital mental retardation. It will be a heavy spirit and financial burden for the society and family. Establishing a noninvasive prenatal diagnosic method becomes very important. The research proposed a method to detect DOWN'S syndrome through the quantitative pyrosequencing of the heterozygous SNPs on placental mRNA of chromosome 21 from maternal plasma. From the reference paper, three placental mRNAs (PLAC4, COL6A2, COL6A1) were selected. An improved allele-specific-amplification was used to screen heterozygous SNPs on the three genes of the chromosome 21 from 84 normal samples. By genotyping 84 genomic DNA samples from normal Chinese population, six SNPs with a relatively high level of heterozygosity were screened out. They are rs8130833, rs4818219 (PLAC4 mRNA), rs2839110, rs1042917, rs35548026(COL6A2 mRNA); rs1053315(COL6A1 mRNA). Heterozygote coverage of 92.9 % was achieved by using a panel of six SNPs on the chromosome 21. Pyrosequencing was used to quantitatively determine the ratio between two alleles of a heterozygote; and the diagnosis of DOWN'S syndrome was thus carried out based on the ratio. Ten clinical samples from DOWN's syndrome patients were quantitatively determined by pyrosequencing, and nine samples were accurately diagnosed by comparing the ratio of the two alleles. The pyrosequencing results showed that the ratio of two alleles were 2:1 or 1:2 for the DOWN's syndrome patients. A key factor which determines the usefulness of the placental expressed transcript for fetal chromosome dosage assessment lies in the co-expression of the allele of the targeted gene. The genotypes of 3 SNPs from the three genes on DNA and RNA from 20 placental tissues were matched with each other based on the results of allele-specific-amplification and pyrosequncing. DNA and RNA have the same genotypes, and the ratio of heterozygsities on RNA is all 1:1. 15 maternal plasma samples from pregnant individuals were determined through the method. Four of the six SNPs involved homozygous mothers and heterozygous fetuses. It suggested that PLAC4 mRNA and COL6A2 mRNA were pregnancy-specific transcripts in maternal plasma. The method has the advantage of low cost, simple process, and time-saving operation and could be potentially applicable to the rapid diagnosis of DOWN'S syndrome.