The analysis of circulating nucleic acids has revealed applications in the noninvasive diagnosis, monitoring, and prognostication of many clinical conditions. Circulating fetal-specific sequences have been detected and constitute a fraction of the total DNA in maternal plasma. The diagnostic reliability of circulating DNA analysis depends on the fractional concentration of the targeted sequence, the analytical sensitivity, and the specificity. The robust discrimination of single-nucleotide differences between circulating DNA species is technically challenging and demands the adoption of highly sensitive and specific analytical systems. We have developed a method based on single-allele base extension reaction and MS, which allows for the reliable detection of fetal-specific alleles, including point mutations and single-nucleotide polymorphisms, in maternal plasma. The approach was applied to exclude the fetal inheritance of the four most common Southeast Asian ␤-thalassemia mutations in at-risk pregnancies between weeks 7 and 21 of gestation. Fetal genotypes were correctly predicted in all cases studied. Fetal haplotype analysis based on a single-nucleotide polymorphism linked to the ␤-globin locus, HBB, in maternal plasma also was achieved. Consequently, noninvasive prenatal diagnosis in a mother and father carrying identical ␤-thalassemia mutations was accomplished. These advances will help in catalyzing the clinical applications of fetal nucleic acids in maternal plasma. This analytical approach also will have implications for many other applications of circulating nucleic acids in areas such as oncology and transplantation. R ecently, much interest has been focused on the biology and diagnostic applications of nucleic acids that are present in the plasma and serum of humans (1, 2). In particular, fetal DNA has been found to exist in maternal plasma (3). This discovery has facilitated the development of noninvasive prenatal diagnostic approaches based simply on the analysis of a maternal blood sample (4). The noninvasive nature of maternal plasmabased approaches represents a major advantage over conventional methods of prenatal diagnosis, such as amniocentesis and chorionic villus sampling, which are associated with a small but finite risk of fetal loss. However, a technical challenge experienced by many workers in the field relates to the ability to discriminate fetal DNA from the coexisting background of maternal DNA in maternal plasma. During pregnancy, fetal DNA amounts to Ϸ3-6% of the total DNA in maternal plasma (5). Hence, the diagnostic reliability of fetal DNA analysis in maternal plasma depends on the sensitivity and specificity of the analytical system for the detection of fetal-specific markers.Fetal SRY and RHD DNA detection from maternal plasma has reached close to 100% accuracy, as confirmed by many largescale evaluations (6-9). The high level of diagnostic accuracy is attained by the analytical sensitivity contributed by the use of real-time quantitative PCR (5, 10) and the analytical specifici...

Molecular characterization of the compound heterozygous condition – ^Gγ(^Aγδβ)^o/β-thalassemia – in four families showing mild β-thalassemia intermedia was carried out using DNA amplification techniques. Using the Amplification Refractory Mutation System (ARMS) to confirm the β-mutations and DNA amplification to detect the 100-kb Chinese-specific ^Gγ(^Aγδβ)^o-deletion, two families were confirmed to possess ^Gγ(^Aγδβ)^o/β-thalassemia with the IVSII No. 654 β⁺-allele. In the third family, the ^Gγ(^Aγδβ)^o-deletion was confirmed in the father and the mother was a β-thalassemia carrier with the cd 41–42 β^o-allele. Their affected child with ^Gγ(^Aγδβ)^o/β-thalassemia was found to be transfusion dependent. The same ^Gγ(^Aγδβ)^o-deletion and β-thalassemia (cd 41–42) was also confirmed in a fourth family. In addition, the mother was also diagnosed with Hb H disease (genotype -α^3.7/–^SEA). Both the children were found to possess ^Gγ(^Aγδβ)^o/β-thalassemia but they were not transfusion dependent and this could be due to co-inheritance of α-thalassemia-2 (genotype-α^3.7/αα) in the children together with their compound heterozygous condition.

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