大规模并行基因组测序技术应用于无创产前诊断染色体非整倍体的研究

目的利用大规模并行基因组测序技术检测孕妇外周血浆中的游离DNA,行胎儿染色体非整倍体的无创产前诊断。方法选择2009年1月到2010年7月在深圳市人民医院产前诊断中心就诊,孕龄在8～30周之间高龄妊娠、唐氏综合征生化筛查高风险和(或)彩超显示胎儿异常等同意介入产前诊断的孕妇941例,抽取孕妇外周静脉血,提取血浆DNA,制备测序文库,应用Illumina HiSeq2000高通量基因测序仪检测,测得的基因序列与人类的参考基因组比对并作统计分析。同时采集胎儿羊水或脐血,经细胞培养后行羊水或脐血细胞染色体核型分析确定染色体非整倍体。结果①941例孕妇血浆样本处理后经大规模并行基因组测序技术检测判定胎儿为唐氏综合征高风险共27例,非唐氏综合征914例;以羊水或脐血染色体核型分析的结果为金标准进行结果对照,检测出的27例唐氏综合征高风险中2例误诊,其中一例核型为47,XXY,一例核型分析正常。经统计分析胎儿唐氏综合征的检出率为100%,检出正确率99.78%,误诊率0.22%;②941例样本中,检出18-三体高风险14例,18-三体低风险927例。与染色体核型分析相比,统计分析显示无创产前基因检测18-三体胎儿的检出率为93.33%(14/15),漏诊率为6.67%(1/15)、误诊率为0。结论利用大规模并行基因组测序技术检测孕妇外周血浆中的游离DNA行无创产前诊断胎儿染色体非整倍体,其敏感性、特异性与染色体核型分析技术具有较高的一致性。该技术具有无创性、高准确性、高通量等优势,具有临床实际应用价值。

Non-invasive Prenatal Diagnosis of Chromosome Aneuploidy by Massively Parallel Genomic Sequencing Technology

Objective To perform non-invasive prenatal diagnosis of chromosome aneuploidy by detecting free DNA in maternal peripheral plasma by using massively parallel genomic sequencing technology.Methods 941 older pregnant women with gestational age among 8 to 30 weeks were chosen,who were registered in prenatal diagnostic centers of Shenzhen People’s Hospital from January 2009 to July 2010,had high-risk in Down syndrome biochemical screening and/or were shown fetal abnormalities by color Doppler ultrasound.The peripheral venous blood from the pregnant women was drawn,plasma DNA was extracted,and the sequencing library was prepared.By using Illumina HiSeq2000,high-throughput sequencing procedure was carried out.The sequencing data were compared with the human reference gene-database and statistically analyzed.Simultaneously,the cells isolated from the fetal amniotic fluid or umbilical cord blood were cultured,and chromosomal karyotyping was done to identify the chromosome aneuploidy.Results ①In 27 out of 941 pregnant women,massively parallel genomic sequencing technology revealed high-risk of Down syndrome.Using the results of chromosomal karyotype analysis of cells from amniotic fluid or umbilical cord blood as the gold standard,2 from the 27 high-risk fetuses were misdiagnosed:one had karyotype of 47,XXY,and the other had normal karyotype.The detection rate of fetal Down syndrome was 100%(25/25)with accuracy rate being 99.78%(939/941),and misdiagnosis rate being 0.22%(2/941);②Of 941 samples,high-risk of 18-trisomy was detected in 14 cases.One 18-trisomy fetus was missed.Compared with karyotyping results,non-invasive prenatal genetic detection rate of 18-trisomy fetus was 93.33%(14/15),missed diagnosis rate was 6.67%(1/15),and misdiagnosis rate was 0.Conclusion Massively parallel sequencing technology for noninvasive prenatal diagnosis of the fetus chromosome aneuploidy by detecting plasma free DNA in pregnant women,which is highly consistent with karyotype analysis in sensitivity and specificity,has great advantages of non-invasion,high-accuracy and high-throughput in clinical application.