title: Prenatal diagnosis of trisomy 21 by quantitatively pyrosequencing heterozygotes using amniotic fluid as starting material of PCR.
abstract: Allelic ratio of an SNP has been used for prenatal diagnosis of fetal trisomy 21 by MALDI-TOF mass spectrometry (MS). Because MALDI-TOF MS is challenging in quantification performance, pyrosequencing was proposed to replace MS by better quantification of allelic ratios. To achieve a simple and rapid clinical diagnostic, PCR with "HpH Buffer" (a buffer with a high pH) was developed to directly amplify amniotic fluid. By the established assay, 114 samples of amniotic fluid were analyzed by pyrosequencing five SNPs of each sample; the allelic ratios of euploid heterozygotes were thus calculated to determine the cut-off values for prenatal diagnosis of trisomy 21. The panel of five SNPs were high in heterozygosity so that at least one heterozygote was found in each sample, and 86% of the samples had at least two heterozygotes, giving a nearly 100% sensitivity (population coverage) of the assay. By using the cut-off values of each SNP, 20 pre-diagnosed clinical samples were detected as trisomy 21 carriers with a confidence level over 99%, indicating that our method and karyotyping analysis were consistent in results. In conclusion, this pyrosequencing-based approach, coupled with direct amplification of amniotic fluid, is accurate in quantitative genotyping and simple in operation. We believe that the approach could be a promising alternative to karyotyping analysis in prenatal diagnosis.
authors: Department of Pharmacology, Jinling Hospital, Nanjing University School of Medicine, Nanjing, 210002, PR China.
authors: Hui
authors: H
authors: Ye
authors: Haiping
authors: H
authors: Wu
authors: Huan
authors: H
authors: Huang
authors: Yunlong
authors: Y
authors: Liu
authors: Bingjie
authors: B
authors: Zou
authors: Lizhou
authors: L
authors: Sun
authors: Guohua
authors: G
authors: Zhou
mesh_terms: D000653:Amniotic Fluid / Q000166:cytology*; D001483:Base Sequence; D002891:Chromosomes, Human, Pair 21 / Q000235:genetics; D004314:Down Syndrome / Q000175:diagnosis* / Q000235:genetics; D005260:Female; D005838:Genotype; D006579:Heterozygote; D006801:Humans; D007621:Karyotyping; D016133:Polymerase Chain Reaction; D020641:Polymorphism, Single Nucleotide / Q000235:genetics; D011247:Pregnancy; D011296:Prenatal Diagnosis / Q000379:methods*; D017422:Sequence Analysis, DNA