Hromadnikova Ilona, Vechetova Lenka, Vesela Klara, Benesova Blanka, Doucha Jindrich, Kulovany Eduard, Vlk Radovan
2nd Clinic of Paediatrics, 2nd Medical Faculty, Charles University, University Hospital Motol, Prague, Czech Republic.
Fetal Diagn Ther. 2005 Jul-Aug;20(4):275-80. doi: 10.1159/000085085.
In this prospective study, we assessed the feasibility of foetal RHD genotyping by analysis of DNA extracted from plasma samples of Rhesus (Rh) D-negative pregnant women using real-time PCR and primers and probes targeted toward exon 7 and 10 of RHD gene.
We analysed 24 RhD-negative pregnant woman and 4 patients with weak D phenotypes at a gestational age ranging from 11th to 38th week of gestation and correlated the results with serological analysis of cord blood after the delivery.
Non-invasive prenatal foetal RHD exon 7 genotyping analyses of maternal plasma samples was in complete concordance with the serological analysis of cord blood in all 24 RhD-negative pregnant women delivering 12 RhD-positive and 12 RhD-negative newborns. RHD exon-10-specific PCR amplicons were not detected in 2 out of 12 studied plasma samples from women bearing RhD-positive foetus, despite the positive amplification in RHD exon 7 region observed in all cases. In 1 case red cell serology of cord blood revealed that the mother had D-C-E-c+e+ C(w)- and the infant D+C-E-c+e+ C(w)+ phenotypes. RhD exon 10 real-time PCR analysis of cord blood was also negative. These findings may reflect that DC(w)- paternally inherited haplotype probably possesses no RHD exon 10. In another case no cord blood sample has been available for additional studies. The specificity of both RHD exon 7 and 10 systems approached 100% since no RhD-positive signals were detected in women currently pregnant with RhD-negative foetus (n = 8). Using real-time PCR and DNA isolated from maternal plasma, we easily differentiated pregnant woman whose RBCs had a weak D phenotype (n = 4) from truly RhD-negative patients since the threshold cycle (C(T)) for RHD exon 10 or 7 amplicons reached nearly the same value like C(T) for control beta-globin gene amplicons detecting the total DNA present in maternal plasma. However in these cases foetal RhD status cannot be determined.
Prediction offoetal RhD status from maternal plasma is highly accurate and enables implementation into clinical routine. We suggest that safe non-invasive prenatal foetal RHD genotyping using maternal plasma should involve the amplification of at least two RHD-specific products.
在这项前瞻性研究中，我们通过使用实时聚合酶链反应（PCR）以及针对RHD基因第7和第10外显子的引物和探针，分析从恒河猴（Rh）D阴性孕妇血浆样本中提取的DNA，评估胎儿RHD基因分型的可行性。
我们分析了24名RhD阴性孕妇和4名弱D表型患者，其孕周在妊娠第11周至38周之间，并将结果与分娩后脐带血的血清学分析结果进行关联。
对所有24名分娩出12名RhD阳性和12名RhD阴性新生儿的RhD阴性孕妇的母血血浆样本进行非侵入性产前胎儿RHD第7外显子基因分型分析，结果与脐带血血清学分析完全一致。在12名怀有RhD阳性胎儿的女性所研究的血浆样本中，有2份未检测到RHD第10外显子特异性PCR扩增产物，尽管所有病例中在RHD第7外显子区域均观察到阳性扩增。在1例中，脐带血红细胞血清学显示母亲为D-C-E-c+e+ C(w)-，婴儿为D+C-E-c+e+ C(w)+表型。脐带血的RhD第10外显子实时PCR分析也为阴性。这些发现可能反映出父系遗传的DC(w)-单倍型可能不具有RHD第10外显子。在另一例中，没有可用的脐带血样本进行进一步研究。由于在目前怀有RhD阴性胎儿的女性（n = 8）中未检测到RhD阳性信号，RHD第7和第10外显子系统的特异性均接近100%。使用实时PCR和从母血血浆中分离的DNA，我们能够轻松区分红细胞具有弱D表型的孕妇（n = 4）与真正的RhD阴性患者，因为RHD第10或第7外显子扩增产物的阈值循环（C(T)）与检测母血血浆中总DNA的对照β-珠蛋白基因扩增产物的C(T)几乎相同。然而，在这些情况下无法确定胎儿的RhD状态。
从母血血浆预测胎儿RhD状态高度准确，可应用于临床常规。我们建议，使用母血血浆进行安全的非侵入性产前胎儿RHD基因分型应至少扩增两种RHD特异性产物。
