新一代测序技术用于胎儿染色体非整倍体无创产前检测的研究

Application of next-generation DNA sequencing for prenatal testing of fetal chromosomal aneuploidies＂

目的探讨新型无创产前检测技术在胎儿染色体非整倍体检测中的应用价值。方法2011年4月19日至2013年12月31日在湖南省妇幼保健院接受孕妇外周血中胎儿游离DNA检测者共4004份，均为单胎，孕周12～35＋5周，根据就诊原因分为唐氏综合征高危组、高龄组及其他原因组3组。提取孕妇外周血，进行血浆分离后提取胎儿游离DNA序列进行高通量测序分析，检测结果阳性者通过羊水穿刺或脐带血穿刺获取胎儿细胞，进行染色体核型分析对结果加以验证，对结果为阴性者进行电话随访，以了解胎儿出生后情况。结果共计4003份完成了胎儿游离DNA染色体非整倍体检测，检测结果提示1．65％（66／4003）存在染色体异常；1份因游离DNA浓度低未达到检测标准而失败。（1）唐筛高危组2568份样本中检出21-三体22例，18-三体3例，13-三体1例，45，X8例，其他染色体异常2例；高龄组1200份样本中检出21-三体13例，18-三体2例，13-三体1例，45，X5例，47，XXN2例，其他染色体异常1例；其他原因组235份样本中检测到21-三体、18-三体、45，x各1例，3例47，XXN；（2）55份样本进行了羊水或（和）脐带血产前诊断。30例21一三体阳性者与4例18-三体阳性者进行了产前诊断，核型均相符，符合率达100％。13例检测结果提示45，x者产前诊断提示3例核型为45，X，2例为45，X／46，XN嵌合体，其余8例核型为46，XN（假阳性）。5例47，XXN检测阳性者中2例产前诊断结果为47，XXN，其余3例为46，XN（假阳性）。另外3例其他染色体异常者经产前诊断，其核型均为46，XN（假阳性）；（3）无创DNA检测结果阴性者共3937份样本。截止至2014年5月，电话随访已出生的新生儿中仅有1例面容异常伴生长发育迟缓，经过外周血核型分析及荧光原位杂交检测确诊为46，XY，rec（14）dup（14q）inv（14）（p12q14）pat。结论胎儿染色体非整倍体无创检测技术具有较高灵敏度及准确度，能有效减少侵入性损害，可以作为传统产前诊断技术的辅助手段。但对于检测13、18、21号染色体以外的其他染色体非整倍体性改变，该技术的灵敏度及准确性还有待进一步的提升。

Objective To explore the value of next-generation sequencing for the non-invasive prenatal testing of fetal chromosomal aneuploidies. Methods Plasma from 4004 women with singleton pregnancy at a gestational age between 12~35＋5 weeks was collected prior to amniocentesis between April 19th 2011 and December 31st 2013. The samples were divided into three groups.. （1） High risk for Down syndrome by biochemical screening; （2） Advanced maternal age; （3） Abnormalities by ultrasound or other methods. Plasma DNA extracted from above samples was sequenced at low coverage. Positive results were verified against the karyotypes of the fetuses. For those with negative results, the fetuses were followed up by telephone call for at least six months after birth. Results Among 4003 samples subjected to non-invasive prenatal diagnosis, 66 （1.65%） had a positive result. In group 1, 22 cases of trisomy 21 （T21）, 3 cases of trisomy 18 （T18）, 1 case of 13 trisomy （T13）, 8 cases of 45, X and 2 cases of other chromosomal abnormality were detected. In group 2, 13 cases of T21, 2 cases of T18, 1 case of T13, 5 cases of 45,X, 2 cases of 47,XXN and 1 case of other chromosomal abnormality were detected. In group 3, 1 case of T21, 1 case of T18, 1 case of T13, and 3 cases of 47,XXN were detected. For 55 samples underwent prenatal diagnosis, 30 cases of T21 and 4 cases of T18 were discovered, which was consistent with the results of non- invasive prenatal diagnosis. For the 13 cases indicated as 45,X, 3 were verified by karyotype analysis, 2 were verified as mosaicism （45,X/46,XN）, 8 were 46, XN （false positives）. For the 5 cases indicated as 47 ,XXN, 2 were verified by karyotype analysis, the other 3 were 46 ,XN （false positives）. Karyotypes of 3 cases suspected for other chromosomal abnormalities were all verified as 46 ,XN （false positive）. Until May 1st 2014, telephone follow-up for those with negative screening results only identified a boy with facial abnormalities and developmental delay, which was similar to his older sister, combined karyotyping and fluorescence in situ hybridization analysis has verified the karyotype of the boy as 46, XY, rec（14）dup（14q） inv（14） （p12q14）pat. Conclusion Our results indicated that sequencing of plasma free I）NA can rapidly detect fetal chromosomal aneuploidies. The method is non-invasive, and the results are highly consistent with karyotype analysis in terms of accuracy and specificity. Non-invasive testing can be used as an effective adjunct to conventional prenatal diagnostic methods, which can greatly reduce unnecessary invasive prenatal diagnosis. However, the sensitivity and accuracy for aneuploidy detection other than chromosome 13/18/21 still need to be improved.