| BackgroundDown syndrome, also known as trisomy 21, is the most common diseases caused by chromosomal abnormalities. It is the most common genetic factors of congenital mental retardation. The most significant and serious clinical manifestation of patients with Down syndrome is moderate to severe mental retardation. Its incidence in live births is 1/600-1/800, and in the total pregnancy is 1/150. Currently, there is no effective treatment for the disease. If a baby were born with Down syndrome, it would bring a heavy financial and emotional burden to the family and society. Therefore, prenatal diagnosis of the disease is important for preventing the live birth of babies with Down syndrome.Karyotype analysis is the gold standard for diagnosis of Down syndrome, and also is the most important diagnostic method for Down syndrome in the international community. But the method is cumbersome and professional. Because of the need of cell culture, diagnostic cycle is long. Generally, from the samples collected to results got, it needs 2-3 weeks. And it is not suitable for scaling up. Therefore, many scholars raised other methods for rapid prenatal diagnosis of Down syndrome, such as fluorescence in situ hybridization, quantitative PCR, gene quantitative analysis technique. Although these techniques can achieve rapid diagnosis, they also have defects, such as the high cost of operation, equipment limitations. It is difficult to use them generally. Therefore, to reduce the birth of live births with Down syndrome and to achieve the purpose of eugenics, it is necessary to study a rapid, simple, effective and economical method of prenatal diagnosis of Down syndrome. Since pyrosequencing technology been reported in 1998, it has been widely used in SNP detection, rapid identification of pathogenic microorganisms, gene methylation analysis and so on. The pyrosequencing technology of our laboratory has been very mature.ObjectiveTo apply quantitative pyrosequencing of heterozygous SNP for rapid prenatal diagnosis of Down syndrome and to investigate its values in clinical application.MethodsSelect 6 SNP loci of rs1053315, rs818219, rs2839110, rs1042917, rs35548026, rs8130833 in the PLAC4, COL6A2, COL6A1 gene on chromosome 21 from reference. To establish heterozygous SNP allele ratio threshold for the diagnosis of Down syndrome , apply the 6 SNP loci to 10 cases of normal people and 5 cases of patients with Down syndrome. Their peripheral blood samples were detected. Then according to the established threshold, 40 cases of prenatal diagnosis(35 cases of amniotic fluid and 5 cases of cord blood) were diagnosed by quantitative pyrosequencing, and were verified by chromosome karyotype analysis.Results(1) The established threshold of allele ratio: 1.0:1.0-1.3:1.0,the case can be diagnosed as diploid chromosome 21; 1.6:1.0-2.2:1.0, the case can be diagnosed as Down syndrome; 1.3:1.0-1.6:1.0 or 6 loci are homozygous, the case can not be diagnosed.(2) Quantitative pyrosequencing analysis: 27 cases were fetal diploid chromosome 21; 7 cases were fetuses with Down syndrome; 5 cases were 6 homozygous SNP loci, so the diagnosis can not be obtained; 1 case failed because of PCR amplification. The results were obtained within 6-8 hours. 8 samples can be detected simultaneously with a portable pyrosequencing instrument. (3) Chromosome karyotype analysis: 33 cases were fetal diploid chromosome 21 and 7 cases were fetuses with Down syndrome. (4) To pyrosequencing, the detection rate was 85% (34/40) and the accuracy was 100% (34/34). Of these, the detection rate and the accuracy of cases with heterozygous SNP were all 100% (34/34).ConclusionThe application of quantitative pyrosequencing of heterozygous SNP for prenatal diagnosis of Down syndrome is rapid, simple, high detection rate and good accuracy. It can be used to clinical rapid prenatal diagnosis of Down syndrome. |
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