摘要：目的 探讨无创产前检测和FISH技术在胎儿染色体非整倍体检测中的临床应用价值。方法 选择2016年10月-2017年10月自愿参与NIPT检测孕妇4807例, 对NIPT检测结果高风险孕妇行羊膜腔穿刺术, 应用染色体核型分析和FISH分析对NIPT结果进行验证, 对NIPT检测结果低风险者进行常规随访。结果 4787例孕妇共检出65例 (1.34%) 染色体数目异常高风险胎儿, 包括1例13-三体、8例18-三体、18例21-三体, 38例性染色体异常。59例孕妇接受进一步的羊膜腔穿刺术及产前诊断, 穿刺率为1.23% (60/4787) 。介入性产前诊断确诊1例13-三体、6例18-三体、16例21-三体和13例性染色体异常胎儿, 阳性预测值分别是:100%、85.7%、88.9%和39.4%。NIPT检测结果提示2例21-三体嵌合体和1例18-三体嵌合体, 利用FISH技术检测均证实为低比例嵌合体。其中345例唐氏筛查高风险孕妇, NIPT检测出6例高风险孕妇经产前诊断确诊, 其余为低风险孕妇通过常规随访得到验证。结论 无创产前基因检测在胎儿染色体非整倍体疾病检测中具有很高的灵敏度、特异性, 联合FISH技术可以快速准确确诊染色体非整倍体, 值得临床应用推广, 降低先天性缺陷儿出生率。
关键词：无创产前检测; 染色体非整倍体; 染色体核型分析; FISH技术;
The clinical application of non-invasive prenatal testing and FISH
TANG Jia SUN Shu-xiang XU Jin-mei LI Qing SUN Tie-lan LI qiu-li YANG Jin-ju LU Ye LI Xiao-min ZHAO Shao-cui LING Ying-cong TAN Wei-lan LI Ling MO Zhong-you ZENG Qin-long
Medical Genetics Center, Jiangmen Maternity and Child health Care Hospital Department of Medical Imaging Center, The First Affiliated Hospital of Jinan University
Abstract：
Objective:To determine the clinical value of non-invasive prenatal test (NIPT) and Fluorescence in situ hybridization (FISH) for the diagnosis of Fetal Chromosoma I Aneuploidy. Methods:From October 2016 to October 2017, 4807 pregnant women volunteered to participate in the study. Amniocentesis was performed on high-risk pregnant women following NIPT. NIPT results were verified by karyotype analysis and FISH analysis. Low-risk patients were routinely followed up after birth. Results:65 of 4787 cases were in high-risk fetal abnormal chromosome aneuploidy (1.34%) , including 1 case of 13-trisomy, 8 cases of 18-trisomy, 18 cases of 21-trisomy, and 38 cases of sex chromosome abnormalities. 59 cases underwent amniocentesis and prenatal diagnosis. The puncture rate was 1.23% (59/4787) . 1 with trisomy 13, 6 with trisomy 18, 16 with trisomy 21 and 13 cases of fetuses with abnormal sex chromosomes were validated by prenatal diagnosis. The positive predictive values were:100%, 85.7%, 88.9%, and 39.4%. The result of NIPT test showed that 2 with mosaic trisomy 21 and 1 with mosaic trisomy 18 were confirmed by FISH. 345 pregnant women with high-risk following serum screening, of these 6 fetal aneuploidy cases were diagnosed by prenatal diagnosis, and the remaining low-risk pregnant women were verified by routine follow-up. Conclusion:Non-invasive prenatal genetic testing has high sensitivity and specificity in the detection of fetal aneuploidy, and combined with FISH can quickly and accurately diagnose aneuploidy of chromosomes. It is worthy of clinical application and promotion to reduce the birth rate of congenital defects.
Keyword：
Non-invasive prenatal testing; Fetal aneuploidy; Karyotype analysis; FISH;
我国出生缺陷总发生率约1.1%, 每年出生的先天残疾儿高达80-120万人, 约占每年出生人口总数4%-6%[1], 其中染色体异常是最为严重的出生缺陷疾病。染色体非整倍体是最常见的染色体疾病, 主要包括21-三体综合征、18-三体综合征、13-三体综合征和性染色体非整倍体, 21-三体、18-三体约占围生期出生缺陷的0.15%, 约占占围生期染色体非整倍体疾病95%[2]。目前针对染色体非整倍体的筛查方法是血清学检测, 该方法只能检测18-三体综合征、21-三体综合征, 其检出率大约70%, 并且假阳性率在5%以上[3], 虽然联合B超及早、中孕筛查可以提高检出率, 但还是不能满足临床需求。而有创性产前诊断进行胎儿细胞培养和染色体分析, 检测周期长, 存在一定的流产风险[4]。
无创产前检测技术 (NIPT, Non-invasive Prenatal Testing) 是一种利用大规模平行测序进行胎儿染色体非整倍体的检测技术, 其特点是无创、灵敏度和特异性高、检测周期短, 弥补了传统检测方法的缺陷, 目前广泛应用于胎儿染色体非整倍体的检测。研究表明13-三体、18-三体和21-三体总体的检测灵敏度和特异性均大于99%[5,6,7]。本研究对4787例接受NIPT的病例进行回顾性分析, 对阳性标本利用染色体核型分析, 荧光原位杂交 (Fluorescence in situ hybridization, FISH) 验证, 探讨无创产前检测的临床应用价值。
1 对象与方法
1.1 研究对象
2016年10月至2017年10月在广东省江门地区进行NIPT检测的孕妇, 共4807例, 年龄17~47岁, 孕周12~37w。研究对象均经遗传咨询和签署知情同意书。
1.2 方法
1.2.1 无创产前筛查
用Streck管采集孕妇外周血10ml, 在72h内1600g 4℃离心10min收集上层血浆, 将收集的血浆16000g常温离心10min, 再吸取上层血浆, 分装, -80℃保存。利用磁珠法对1200μl血浆进行DNA提取, 最终洗脱体积为42μl, 使用Qubit3.0测定DNA质量浓度。取全部的DNA溶液进行建库 (PCR-Free) , 包括末端修复、加接头, 再对文库进行Real-time PCR定量, 最后通过Nextseq CN500进行测序, 对测序数据进行生物信息学分析, 计算Z值, 评估胎儿患病风险:Z>3为高风险, -3<Z<3, 则为低风险。电话通知NIPT高风险的孕妇, 来院进行产前咨询, 建议进行羊水穿刺, 进行FISH快速诊断和染色体核型分析确诊。
1.2.2 羊水G显带核型分析
B超引导下行羊膜腔穿刺, 取羊水20 ml, 置于2支无菌离心管中, 离心后取沉淀的羊水细胞分别无菌接种于5 ml羊水培养基, 置于37℃、5%二氧化碳培养箱培养7-9天。收获细胞后行G显带制片并分析核型, 异常核型按《人类细胞遗传学国际命名体制》 (ISCN1995) 进行描述[8]。
1.2.3 羊水FISH检测
取5ml羊水, 采用美国雅培公司试剂盒, CEP 18/X/Y与LSI 13/21双探针设计, 按照说明书标准常规操作流程, 羊水细胞经预处理、杂交和洗涤复染, 置荧光显微镜下判断最后结果, 随机计数100个细胞以上, 48h内出检测报告[9]。
1.2.4 随访
由本院产前诊断中心护士对所有受检孕妇进行电话随访妊娠结局。
2 结果
2.1 4787例孕妇无创产前筛查结果
4807例孕妇有20例因为胎儿DNA浓度低于3.5%, 达不到检测要求检测失败, 本研究失败率约为0.42% (20/487) 。检测成功4787例孕妇通过NIPT检测提示染色体非整倍体高风险65例, 异常检出率为1.34% (65/4787) , 其中59例孕妇接受进一步的羊膜腔穿刺术及羊水细胞染色体核型分析, 穿刺率为1.23% (59/4787) , 5例拒绝侵入性产前诊断 (4例X-和1例X+) , 1例18三体在产前诊断之前因B超检查结果胎儿多发畸形而人工引产。本研究NIPT检测技术对13-三体、18-三体、21-三体和性染色体的阳性预测值分别是:100%、85.7%、88.9%和39.4%。
4787例孕妇中345例为唐氏血清学筛查高风险, 通过NIPT检测提示染色体非整倍体高风险1.74% (6/345) , 包括5例21-三体和1例18-三体, 进行产前诊断 (羊水FISH检测和染色体核型分析) 均确诊为真阳性, NIPT检测低风险的339例唐氏血清学筛查高风险孕妇经产后3个月随访未见无异常。
2.2 羊水G显带核型分析及FISH结果分析
对1例13-三体高风险、7例18-三体高风险、18例21-三体高风险, 33例性染色体异常孕妇进行羊水细胞染色体核型分析, 确诊1例13-三体、6例18-三体、16例21-三体和13例性染色体异常, 详见表1。
表1 NIPT高风险结果与FISH及核型分析结果的比较
Mos:嵌合;“/”:未进行FISH检测
将染色体核型分析结果与FISH检测结果对比分析, FISH技术检测快速, 结果判读直观, FISH技术检测出染色体非整倍体异常与核型分析结果一致, 其中FISH检测出3例嵌合体。NIPT结果提示1例18-三体高风险嵌合体, 2例21-三体高风险嵌合体, 羊水细胞染色体核型验证结果均无异常, 利用FIHS技术检测结果2例21-三体高风险嵌合体, 嵌合比例分别为18%, 9%, 1例18-三体高风险嵌合体, 嵌合比例为6%。 (图1见封三)
2.3 随访结果
4722例NIPT低风险的孕妇, 根据其预产期, 在产后3个月进行随访研究, 随访结果均无异常。
3 讨论
1997年香港中文大学卢煜明教授发现孕妇血浆存在胎儿游离DNA[10], 后续研究证实孕妇血浆中胎儿游离DNA主要来源于胎盘组织, 在孕12w以后可稳定存在, 随着孕周的增长而逐步增加, 其含量约占总游离DNA的10%[11,12]。本研究4787例孕妇有20例因为胎儿DNA浓度低于3.5%, 达不到检测要求, 判断为检测失败。以往研究表明约2%孕妇因为胎儿游离DNA浓度低, 导致NIPT检测失败, 同时限制性胎盘嵌合 (CPM) 1%~2%的发生率, 也可能造成NIPT检测结果不准确[13,14,15,16]。
该研究对345例唐氏血清学筛查高风险孕妇进行NIPT检测, 结果仅有6例阳性, 其余均为阴性, 结果表明唐氏血清学筛查假阳性率较高, 因此利用NIPT技术对唐氏血清学筛查高风险孕妇进行检测, 能有效降低假阳性, 避免产前介入性诊断的流产风险及孕妇心理创伤。
3例NIPT检测结果提示染色体三体高风险嵌合体, 羊水细胞核型和FISHI结果不一致, 因为羊水细胞染色体核型分析需要进行细胞培养, 细胞培养过程发生细胞选择性生长, 而FISH技术不经过细胞培养步骤, 直接检测和观察细胞染色体数量, 能准确直观反映羊水细胞染色体情况[17,18]。
NIPT检测结果提示性染色体异常而进行产前诊断确诊的样本有33例, 而验证结果仅有13例样本为性染色体异常, 并且有3例NIPT检测结果为性染色体单体, 而羊水细胞核型验证结果为性染色体三体或性染色体三体嵌合体。研究表明造成NIPT检测性染色体异常结果的原因与X染色体GC含量偏移, X和Y染色体高度同源不利于分辨有关[19]。也有研究表明8.6%的原因是因为孕妇本身存在性染色体异常[20]。
研究表明胎儿游离DNA浓度越高, NIPT的检出率和准确性越高[21,22]。目前NIPT用于估算胎儿游离DNA浓度的方法主要是通过Y染色体进行估算, 而女胎的胎儿游离DNA浓度计算准确性低问题一直无法有效解决[23]。本研究NIPT检测存在性染色体异常高比例假阳性问题, 可能因为胎儿游离DNA浓度估算不准导致。我们进一步发现性染色体异常的胎儿中, 含有Y染色体的胎儿, 阳性预测值高达80%。本研究还发现准确检测出的3例三体嵌合胎儿, 均含Y染色体, 因此推测胎儿游离DNA的估算准确性直接影响NIPT检测结果的准确性, 通过Y染色体比例能准确估算胎儿游离DNA浓度, 提高NIPT的检测准确性。
4 展望
利用高通量测序技术结合生物信息学分析, 可以检测胎儿游离DNA判断胎儿染色体是否异常, 具有很高的灵敏度、准确性。基于加大测序量和优化生物信息流程的优化NIPT, 还可以检测染色体大片段缺失或重复[24,25]。NIPT不断发展, 逐渐应用于单基因病无创产前检测, 通过父母及先证者的基因组数据构建单倍型, 进行单基因病的产前无创检测[26,27,28,29]。相信随着新技术和算法的不断改进创新, 在不久的将来NIPT可以实现大部分单基因遗传病的无创产前检测, 阻断遗传病的发生, 防止出生缺陷, 推动全球优生优育事业的发展。
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