基于RASSF1A为标志的无损性孕妇血浆中胎儿RHD-DNA检测方法的建立  

Noninvasive fetal RHD genotyping based on RASSF1A gene with fetal DNA from maternal plasma

作　　者：马玲[1] 刘衍春[1] 郑凌[1] 韩树萍[2] 吴敏慧[1] 刘毅[1] 魏鹏[1] 孙俊[1] 

机构地区：[1]江苏省血液中心,江苏南京210042 [2]南京市妇幼保健院

出　　处：《中国输血杂志》2014年第7期696-699,共4页Chinese Journal of Blood Transfusion

摘　　要：目的建立基于表观遗传标志RASSF1A为标志的孕妇外周血浆中胎儿RHD-DNA检测路径,用于无损性产前胎儿RhD血型预测。方法首先采用PCR-SSP对孕妇外周血细胞基因组DNA做RHD基因多个外显子检测,以确定其RHD基因型;其次对无外显子表达或部分外显子表达的孕妇,对其外周血浆中胎儿游离的DNA采用RTPCR扩增其RHD基因exon7与10,同时扩增SRY基因;另对exon 7、10和SRY都为阴性的标本检测甲基化RASSF1A基因作为胎儿DNA存在的标志。待胎儿分娩后,将基因检测结果与婴儿血清学检测结果比对,以评价该方法的准确性。结果 57位受检孕妇中,含RHD全部外显子者有17人;3人含exon 1和10,为RHD1-CE(2-9)-D10杂化基因;37人不含外显子。后两者40名孕妇中,22人检测出SRY基因,出生胎儿均为男性;另有2人为假阴性结果。39人检测出exon 7和10,其中38人基因型结果与胎儿出生后血清学结果相符,1例假阳性结果;1人未检测到SRY、exon 7和10的标本,检测到RASSF1A证明胎儿DNA存在,且胎儿出生后证实为RhD阴性女婴。结论建立了基于RASSFIA为标志的、符合中国人RHD基因特点的RhD阴性孕妇血浆游离DNA中胎儿RHD基因的检测程序,可用于临床无损性产前胎儿RhD血型预测。Objective To design a protocol for non-invasive detection of fetal RhD status by using circulating cell-free fetal DNA from maternal plasma. Methods Detecting maternal RHD status first, those without RHD gene or with partly RHD exons specimens were selected for the further fetal RHD exon7 and exon10 detection. The presence of circulating cell-free fetal DNA （effDNA） was confirmed by testing SRY and RASSF1A. The real-time PCR results were ascertained by blood typing of the neonate at birth. Results Among 57 RhD-negative pregnant women,17 samples whioh had all the RHD exons were detected; 3 samples were RHD1-CE（2-9）-D10; 37 samples had null RHD exons. For the latter two, total 40 specimens, 22 cases were detected SRY, which correlated correctly with delivery of male infants. There were also two false-negative results. 39 samples had the specifically amplified RHD exon7 and exon 10 with one false-positive. The presence of cffDNA for the remaining one sample with neither RHD exons nor SRY signals was verified by detection of the fetal-associated hypermethylated RASSF1A gene after BstUI digestion. Conclusion The proposed protocol of detection fetal RHD by using fetal DNA from maternal plasma is specially considering RHD gene character of Chinese population and it can be used for noninvasive prenatal diagnosis of fetal RhD blood type.

关 键 词：血浆游离胎儿DNA RHD基因型分型 无损性 RHD阴性 表观遗传学标志 RASSF1A 

分 类 号：R457.11[医药卫生—治疗学] R446.112[医药卫生—临床医学]