孕妇外周血中游离胎儿DNA检测在诊断胎儿染色体异常中的应用价值

Value of detection of cell-free fetal DNA in maternal plasma in the prenatal diagnosis of chromosomal abnormalities

目的 探讨孕妇外周血中游离胎儿DNA检测在诊断胎儿染色体异常中的应用价值.方法 2011年4月1日至2012年5月30日在解放军总医院接受孕妇外周血中游离胎儿DNA检测者3200例,均为单胎,孕周（20.3±3.8）周,按照孕妇年龄及血清学筛查结果分为3组：（1）唐氏综合征筛查（唐筛）高危组：1720例,预产年龄＜35岁,唐氏综合征血清学筛查提示21三体高风险; （2）高龄组：1310例,预产年龄≥35岁;（3）其他原因组：170例,预产年龄＜35岁,未行唐筛,或唐筛结果为低风险.唐筛高危组及高龄组共计 3030例孕妇均因担心穿刺手术的流产风险或产前检查医院无产前诊断条件等原因,未行介入性产前诊断.由深圳华大基因临床检验中心采用Illumina HiSeq 2000测序平台对孕妇外周血中游离胎儿DNA进行序列分析,对检测结果阳性者进行介入穿刺及胎儿染色体核型分析;对检测结果阴性者行电话随访其胎儿出生后情况.结果（1）3组孕妇共计3200例均成功完成游离胎儿DNA检测,检测结果为阳性者共31例,包括21三体27例,18三体4例.其中唐筛高危组检出21三体16例,18三体1例;高龄组检出21三体7例,18三体2例;其他原因组检出21三体4例,18 三体1例.（2）游离胎儿DNA检测结果阳性的31例孕妇中,26例（84％）进行了羊膜腔穿刺及染色体核型分析.27例21三体检测阳性者中有23例 （85％）进行了羊膜腔穿刺,染色体核型均为47,XN,＋21,诊断符合率100％;4例未行核型分析,其中1例是在孕20周前超声检查已提示胎儿存在明显结构异常（室间隔缺损、右位心、脉络丛囊肿）;1例在检测结果回报前已发生胎死宫内;另外2例为外地孕妇,均已分娩过健康后代,此次拒绝行染色体核型分析,在当地医院直接引产.4例18三体检测阳性者中有3例进行了羊膜腔穿刺,其中2例核型为47,XN,＋18;另l例为低比例嵌合,核型为 46,XN/47,XN,＋18,18三体细胞占羊水总细胞的2％,未行引产,胎儿出生后未发现结构异常;有l例因在穿刺手术前已发生胎死宫内,未能进行核型验证,其前超声提示胎儿较孕周小3周,且全身水肿.（3）3组孕妇血浆中游离胎儿DNA检测结果阴性者3173例,经电话随访,截止至2012年5月 30日,已有1230例新生儿出生,经检查均未发现唐氏综合征患儿.结论 游离胎儿DNA检测是一种安全、准确、高通量的21三体产前检测方法,与染色体核型分析结果相符;作为唐氏综合征患儿血清学筛查高风险孕妇的进一步筛选方法,可大幅度减少介入性产前诊断,并可作为临床诊断唐氏综合征的依据.

Objective To investigate the value of detection of fetal cell-free fetal DNA（cff-DNA）in maternal plasma in the prenatal diagnosis of chromosomal abnormalities.Methods The plasma from 3200 gravidas（singleton with 20.3 ± 3.8 gestational weeks）was collected from April 1st 2011 to May 30th 2012.They were divided into 3 groups：（1）To tally 1720 cases were included in the high-risk serological screening group,in which women were younger than 35 years and got high-risk results in serological screening;（2）To tally 1310 cases were included in the advanced age group,in which women＇s age was more than 35 years;（3）To tally 170 cases were included in the supplementary group,in which women were younger than 35 years and got low-risk results in serological screening,or women who didn＇t take serological screening tests.All the 3030 gravidas in group 1 and 2 didn＇t take invasive prenatal diagnosis because of fear of abortion or short of prenatal diagnosis.Cff-DNA were detected by next generation sequencing in Shenzhen BGI Genomics Center for clinical laboratory.Amniocentesis and karyotype analysis were provided to the positive cases and women with negative results were followed-up by telephone.Results（1）The 3200 cases took cff-DNA detection,and 31 cases got positive results,including 27 cases of trisomy 21 and 4 cases of trisomy 18.Sixteen cases of trisomy 21 and 1 case of trisomy 18 were in the high-risk serological screening group.7 cases of trisomy 21 and 2 cases of trisomy 18 were in the advanced age group.Four cases of trisomy 21 and 1 case of trisomy 18 were in the supplementary group.（2）And the 84%（26/31）cff-DNA detecting positive cases received amniocentesis.In the 27 trisomy 21 positive cases,23 received amnioeentesis and got karyotype of 47XN,＋ 21,with the diagnostic accordance rate of 100%.In the 4 cases who didn＇t take karyotype analysis,fetal anomaly（ventricular septal defect,dextrocardia and choroid plexus cyst）was found in 1 case before 20 gestational weeks;intrauterine fetal demise happened in 1 case before getting the result;2 other cases who already had healthy children took abortion in the local hospital without taking amniocentesis.In the 4 trisomy 18 positive cases,3 took amniocentesis,2 of which were trisomy 18 and took abortion,the other was chimera（46,XN/47,XN,＋ 18）with only 2% cells of trisomy 18,with no malformation found after delivery.Hypoevolutism（3 weeks less than gestational week）,general hydropsy and intrauterine fetal demise happened before the other case took amniocentesis.（3）Follow up of cff-DNA negative cases：until May 30th 2012,no Down＇s baby was found in the 1230 cases with cff-DNA test negative results.Conclusions（1）The non-invasive fetal trisomy test（NIFTY）by next generation sequencing is a safe,accurate and high throughput method for the prenatal diagnosis of trisomy-21.（2）Use NIFTY as a further screening for pregnant women with high-risk serological screening results could lower invasive prenatal diagnosis rate.（3）Cases with positive NIFTY test results should receive amniocentesis and karyotype analysis to confirm the diagnosis before abortion.