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目的利用孕妇外周血中单个胎儿有核红细胞(NRBC)进行β地中海贫血无创性产前基因诊断,与样本为绒毛、羊水的创伤性产前诊断结果相比较,评估其可靠性及可行性。方法显微操作单个获取孕妇外周血中 NRBC,引物延伸预扩增(PEP)对单个 NRBC 进行全基因组扩增,短串联重复序列(STR)鉴定所取细胞的来源。证实为胎儿细胞的 PEP 产物作为模板进行β珠蛋白基因扩增,反向点杂交确定胎儿β珠蛋白基因型。结果光学显微镜下每例发现 NRBC 4～13(10.64±2.26)个,经 STR 基因型分析鉴定,每例有胎儿 NRBC 2～8(4.64±1.79)个,约43.6%的 NRBC 来源于胎儿。单个 NRBC 经 PEP 后可用于鉴定胎儿β珠蛋白基因点突变类型,与样本为绒毛、羊水的创伤性产前诊断结果相比较,诊断符合率为85.7%。结论凭借 PEP 和反向点杂交技术,利用母血中单个胎儿 NRBC 可进行β地中海贫血产前基因诊断,诊断准确性和可靠性较高,是一条可供尝试的无创性产前诊断途径。
Objective To evaluate the noninvasive prenatal diagnosis of β thalassemia using single fetal nucleated erythrocytes (NRBC) in the peripheral blood of pregnant women and to evaluate its reliability and feasibility compared with the traumatic prenatal diagnosis of villus and amniotic fluid in samples. Methods The NRBC of peripheral blood of pregnant women was obtained by micromanipulation. The whole genome of single NRBC was amplified by primer extension pre-amplification (PEP). The origin of the cells was identified by short tandem repeat (STR). The PEP product confirmed to be fetal cells was used as a template for amplification of the beta globin gene, and reverse dot blot was used to determine the fetal beta globin genotype. Results There were 4 ~ 13 (10.64 ± 2.26) NRBCs in each case under the light microscope. The genotypes of STRs were identified in each case, with 2 to 8 (4.64 ± 1.79) cases of NRBC and about 43.6% of NRBC from the fetus. A single NRBC can be used to identify the type of point mutation in fetal β-globin gene after PEP. Compared with traumatic prenatal diagnosis of villus and amniotic fluid, the coincidence rate of the single NRBC was 85.7%. Conclusion PEP and reverse dot blot hybridization can be used to diagnose β-thalassemia prenatal gene by using single fetus NRBC in maternal blood, which has a high diagnostic accuracy and reliability. It is a promising noninvasive prenatal diagnostic pathway.