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    ABSTRACT: 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.
    Zhonghua er ke za zhi. Chinese journal of pediatrics 01/2008; 45(12):917-21.