Semi-nested Taqman real-time quantitative PCR for noninvasive prenatal diagnosis of Bart's hydrops fetalis.

BACKGROUND: Non-invasive prenatal diagnosis based on detection of fetal cell-free DNA is limited when mother and father are both carriers for the same autosomal recessive mutation. OBJECTIVE: Develop the semi-nested Taqman real-time PCR for quantification of alpha-thalassemia-1 SEA type deletion allele in plasma of alpha-thalassemia-1 SEA carriage pregnancies. MATERIAL AND METHOD: Plasma DNA was extracted from six women who carried fetuses with normal, 11 with heterozygote alpha-thalassemia-1 SEA type deletion and seven with Bart's hydrops fetalis. DNA was amplified using conventional PCR with the primary specific primer set for alpha-thalassemia-1 SEA type deletion. PCR product was then subjected to the semi-nested real-time PCR using the secondary specific primer and Taqman probe set for alpha-thalassemia-1 SEA type deletion. The standard curve was constructed using ten-fold serial dilutions of conventional PCR product of the heterozygote alpha-thalassemia-1 SEA type deletion. RESULTS: Women who carried fetuses with Bart's hydrops fetalis displayed a trend toward higher mean copy number of alpha-thalassemia-1 SEA type deletion allele vs. women who carried fetuses with normal and heterozygote, albeit not reaching statistical significance. CONCLUSION: The maternally inheritedfetal allele present in maternal plasma is difficult to discern the fetal cell-free DNA from a higher background DNA of the mother Thus, further investigation is needed to improve the diagnosis ofBart's hydrops fetalis using this technique.

Analysis of real-time PCR cycle threshold of alpha-thalassemia-1 Southeast Asian type deletion using fetal cell-free DNA in maternal plasma for noninvasive prenatal diagnosis of Bart's hydrops fetalis.

BACKGROUND: Noninvasive prenatal diagnosis based on detection of fetal cell-free DNA is hampered when mother and father are both carriers for the same autosomal recessive mutation. OBJECTIVE: To compare the diagnosis of Bart's hydrops fetalis using conventional Gap-PCR analysis of fetal cells/tissues with the measurement of quantitative difference (deltaCp) between alpha-thalassemia-1 SEA type deletion gene (C(T-mutant)) and wild type alpha-globin gene (C(T-wild type)) in plasma of pregnancies by using the Taqman real-time quantitative PCR. MATERIAL AND METHOD: Plasma DNA samples were collected from three groups of pregnancies whose fetuses have known thalasemia status (7 normal, 11 heterozygote alpha-thalassemia-1 SEA type deletion, and 7 Bart's hydrops fetalis). The alpha-thalassemia-1 SEA type deletion gene and wild type alpha-globin gene were quantified by using Taqman real-time quantitative PCR and then the delta C(T) was analyzed by subtracting the C(T-mutant) from C(T-wild type). RESULTS: Mean deltaC(T) values were not significantly different among the three groups. However, women whose fetuses were diagnosed as Bart's hydrops fetalis had a higher proportion (43%) of plasma DNA samples that had negative deltaC(T) value than women whose fetuses were diagnosed as normal or heterozygote alpha-thalassemia-1 SEA type deletion (0 and 27%, respectively). CONCLUSION: Further investigations are needed to improve the diagnosis of Bart's hydrops fetalis using fetal cell-free DNA.