摘要
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 investigate non-invasive prenatal genetic diagnosis of β-thalassaemia using a single fetal nucleated erythrocyte （NRBC） from maternal blood by comparing with the genotype obtained from chorionic villus or amniocytes, and to evaluate the diagnostic results in reliability and feasibility of this method. Methods Maternal blood samples were obtained from 28 pregnant women at risk of β thalassaemia during 9-34 weeks of gestation. NRBCs in maternal blood were enriched by single density gradient separation, stained with benzidine, and then collected by micromanipulation individually. After primer extension preamplification （PEP） of the entire genome from each single NRBC, short tandem repeat （STR） genotype was analyzed after further amplification of this gene. Single NRBC was tested individually to identify if it was fetal or maternal in origin by STR genotype of NRBC and its corresponding parents. β-globin DNA fragments were amplified with nested-PCR using PEP product of a single fetal NRBC that was determined to be fetal in origin. Fetal β-globin genotypes were analyzed by reverse dot-blot hybridization （RDB）, the accuracy was evaluated by comparing with genotype which had been determined on DNA obtained from chorionic villus （CVS） or amniocytes. Results A total of 298 NRBCs were found in all of 28 pregnant women at a range of 4 to 13 per 5 ml venous blood. After PEP, about 43.6% of NRBCs were determined to be fetal in origin by STR typing. Using PEP product of a single fetal NRBC as template, β- globin DNA fragment was examined on agarose gel after nested-PCR, amplification efficiency was 90.8% （118/130）. Fetal β-globin genotypes were achieved successfully in all cases with RDB. Comparing with the genotypes which were obtained from CVS or amniocytes, the rate of diagnostic accuracy was 85.7% （24/ 28）. Conclusions PEP technique and STR genotype analysis provide effective method for identification of single nucleated erythrocyte from maternal blood in origin. With the tech
出处
《中华儿科杂志》
CAS
CSCD
北大核心
2007年第12期917-921,共5页
Chinese Journal of Pediatrics
基金
广西科学基金资助项目(桂科攻0015042)
关键词
幼红细胞
Β地中海贫血
产前诊断
Erythroblasts
Beta-thalassemia
Prenatal diagnosis
