D'Souza E, Sawant P M, Nadkarni A H, Gorakshakar A, Ghosh K, Colah R B
Department of Haematogenetics, National Institute of Immunohaematology (ICMR), K.E.M. Hospital Campus, Parel, Mumbai, Maharashtra, India.
J Postgrad Med. 2013 Jan-Mar;59(1):15-20. doi: 10.4103/0022-3859.109483.
Prenatal diagnosis of hemoglobinopathies enables couples at risk to have a healthy child. Currently used fetal sampling procedures are invasive with some risk of miscarriage. A non-invasive approach to obtain fetal deoxyribonucleic acid (DNA) for diagnosis would eliminate this risk.
To develop and evaluate a non-invasive prenatal diagnostic approach for hemoglobinopathies using cell-free fetal DNA circulating in the maternal plasma.
Couples referred to us for prenatal diagnosis of hemoglobinopathies where the maternal and paternal mutations were different were included in the study.
Maternal peripheral blood was collected at different periods of gestation before the invasive fetal sampling procedure was done. The blood was centrifuged to isolate the plasma and prepare DNA. A size separation approach was used to isolate fetal DNA. Nested polymerase chain reaction (PCR)-based protocols were developed for detection of the presence or absence of the paternal mutation.
There were 30 couples where the parental mutations were different. Of these, in 14 cases the paternal mutation was absent and in 16 cases it was present in the fetus. Using cell-free fetal DNA from maternal plasma, the absence of the paternal mutation was accurately determined in 12 of the 14 cases and the presence of the paternal mutation was correctly identified in 12 of the 16 cases. Thus, this non-invasive approach gave comparable results to those obtained by the conventional invasive fetal sampling methods in 24 cases giving an accuracy of 80.0%. Although the nested PCR approach enabled amplification of small quantities of cell-free DNA from maternal plasma at different periods of gestation after size separation to eliminate the more abundant maternal DNA, an accurate diagnosis of the presence or absence of the paternal mutation in the fetus was not possible in all cases to make it clinically applicable.
血红蛋白病的产前诊断可使有风险的夫妇生育健康的孩子。目前使用的胎儿采样程序具有侵入性，存在一定的流产风险。一种获取胎儿脱氧核糖核酸（DNA）进行诊断的非侵入性方法将消除这种风险。
开发并评估一种利用母血中循环的游离胎儿DNA对血红蛋白病进行非侵入性产前诊断的方法。
本研究纳入了因血红蛋白病产前诊断前来就诊且父母突变不同的夫妇。
在进行侵入性胎儿采样程序之前的不同孕期采集孕妇外周血。对血液进行离心以分离血浆并制备DNA。采用大小分离方法分离胎儿DNA。开发了基于巢式聚合酶链反应（PCR）的方案来检测父系突变的有无。
有30对夫妇的父母突变不同。其中，14例胎儿不存在父系突变，16例胎儿存在父系突变。利用母血血浆中的游离胎儿DNA，在14例中的12例准确确定了父系突变的缺失，在16例中的12例正确识别了父系突变的存在。因此，这种非侵入性方法在24例中得到的结果与传统侵入性胎儿采样方法相当，准确率为80.0%。尽管巢式PCR方法能够在大小分离后在不同孕期从母血血浆中扩增少量游离DNA以去除更丰富的母系DNA，但并非在所有情况下都能准确诊断胎儿中父系突变的有无，使其无法在临床上应用。
