Wei Hong-ying, Long GuifFang, Lin Wei-xiong, Li Shu-quan
Department of Pediatrics, First Affiliated Hospital, Guangxi Medical University, Nanning 530021, China.
Zhonghua Er Ke Za Zhi. 2007 Dec;45(12):917-21.
To investigate non-invasive prenatal genetic diagnosis of beta-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.
Maternal blood samples were obtained from 28 pregnant women at risk of beta 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. beta-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 beta-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.
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, beta-globin DNA fragment was examined on agarose gel after nested-PCR, amplification efficiency was 90.8% (118/130). Fetal beta-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).
PEP technique and STR genotype analysis provide effective method for identification of single nucleated erythrocyte from maternal blood in origin. With the techniques PEP and RDB, fetal beta-globin genetic diagnosis was achieved using a single fetal NRBC from maternal blood. The method had a high accuracy and reliability in diagnosis. It may become an optional approach to non-invasive prenatal diagnosis of beta-thalassemia.
通过与绒毛膜绒毛或羊水细胞获得的基因型进行比较，研究利用母体血液中的单个胎儿有核红细胞（NRBC）进行β地中海贫血的无创产前基因诊断，并评估该方法诊断结果的可靠性和可行性。
从28例妊娠9 - 34周有β地中海贫血风险的孕妇中采集母体血样。通过单密度梯度分离富集母体血液中的NRBC，用联苯胺染色，然后通过显微操作逐个收集。对每个单个NRBC的全基因组进行引物延伸预扩增（PEP）后，对该基因进一步扩增后分析短串联重复序列（STR）基因型。通过NRBC及其相应父母的STR基因型对单个NRBC进行单独检测，以确定其来源是胎儿还是母体。使用确定为胎儿来源的单个胎儿NRBC的PEP产物，通过巢式PCR扩增β珠蛋白DNA片段。通过反向点杂交（RDB）分析胎儿β珠蛋白基因型，通过与从绒毛膜绒毛（CVS）或羊水细胞获得的DNA上确定的基因型进行比较来评估准确性。
28例孕妇的所有血样中共发现298个NRBC，每5 ml静脉血中4 - 13个不等。PEP后，通过STR分型确定约43.6%的NRBC来源为胎儿。以单个胎儿NRBC的PEP产物为模板，巢式PCR后在琼脂糖凝胶上检测β珠蛋白DNA片段，扩增效率为90.8%（118/130）。所有病例均通过RDB成功获得胎儿β珠蛋白基因型。与从CVS或羊水细胞获得的基因型相比，诊断准确率为85.7%（24/28）。
PEP技术和STR基因型分析为鉴定母体血液中单个有核红细胞的来源提供了有效方法。利用PEP和RDB技术，通过母体血液中的单个胎儿NRBC实现了胎儿β珠蛋白基因诊断。该方法诊断准确性和可靠性高。它可能成为β地中海贫血无创产前诊断的一种可选方法。
