Kacem Narjes, Jemni-Yacoub Saloua, Chiaroni Jacques, Bailly Pascal, Silvy Monique
French Blood Institute-Alpes-Méditerranée, Marseille, France UMR 7268 ADES, Aix-Marseille Université-EFS-CNRS, Marseille, France Regional Blood Transfusion Centre, Research Unit "UR06SP05", Sousse, Tunisia.
Regional Blood Transfusion Centre, Research Unit "UR06SP05", Sousse, Tunisia.
Blood Transfus. 2015 Jan;13(1):59-65. doi: 10.2450/2014.0308-13. Epub 2014 Jun 19.
The choice of a molecular test for first intention determination of paternal RHD zygosity, before entering into invasive diagnostics, is important for the management of pregnancies at risk of haemolytic disease of the foetus and newborn related to anti-RhD.
RHD zygosity was evaluated in 370 RH:1 Tunisian donors by polymerase chain reaction - sequence-specific polymorphism (PCR-SSP) analysis and polymerase chain reaction - restriction fragment length polymorphism (PCR-RFLP) amplification of hybrid Rhesus box and by real time quantitative polymerase chain reaction (RQ-PCR) specific for RHD exon 5. To evaluate the accuracy of molecular tests in the cases of discordant results, the ten exons of RHD and Rhesus boxes were amplified by PCR and sequenced.
Molecular investigations revealed that our 370 donors comprise 193 dizygous and 145 hemizygous individuals and 32 subjects whose zygosity remains unknown. Positive predictive values were higher than 99% for all the methods, reaching 100% for RQ-PCR. Negative predictive values were 83.24%, 87.27% and 98% for PCR-SSP, PCR-RFLP and RQ-PCR respectively. This study also revealed 19 novel Rhesus box polymorphisms and three novel RHD alleles: RHD(Trp185Stop), RHD(Ala176Thr) and RHD(Ile342Ile).
RQ-PCR is the most convenient method for first intention determination of paternal RHD zygosity in Tunisians. However, taking into account positive and negative predictive values, PCR-RFLP could be an alternative despite the heterogeneity of Rhesus boxes and the complexity of RHD.
在进行侵入性诊断之前，选择一种用于初步确定父方RHD合子性的分子检测方法，对于管理有胎儿和新生儿溶血病风险（与抗-RhD相关）的妊娠至关重要。
通过聚合酶链反应-序列特异性多态性（PCR-SSP）分析、杂交恒河猴盒的聚合酶链反应-限制性片段长度多态性（PCR-RFLP）扩增以及针对RHD外显子5的实时定量聚合酶链反应（RQ-PCR），对370名RH:1突尼斯献血者的RHD合子性进行评估。为了评估分子检测在结果不一致情况下的准确性，通过PCR扩增RHD的十个外显子和恒河猴盒并进行测序。
分子研究表明，我们的370名献血者包括合子型个体193例、半合子型个体145例以及32例合子性未知的个体。所有方法的阳性预测值均高于99%，RQ-PCR达到100%。PCR-SSP、PCR-RFLP和RQ-PCR 的阴性预测值分别为83.24%、87.27%和98%。本研究还发现了19种新的恒河猴盒多态性和三种新的RHD等位基因：RHD(Trp185Stop)、RHD(Ala176Thr)和RHD(Ile342Ile)。
RQ-PCR是初步确定突尼斯父方RHD合子性最便捷的方法。然而，考虑到阳性和阴性预测值，尽管恒河猴盒存在异质性且RHD复杂，但PCR-RFLP仍可作为一种替代方法。
