Prenatal diagnosis of monogenic diseases, such as cystic fibrosis and ␤-thalassemia, is currently offered as part of public health programs. However, current methods based on chorionic villus sampling and amniocentesis for obtaining fetal genetic material pose a risk to the fetus. Since the discovery of cell-free fetal DNA in maternal plasma, the noninvasive prenatal assessment of paternally inherited traits or mutations has been achieved. Due to the presence of background maternal DNA, which interferes with the analysis of fetal DNA in maternal plasma, noninvasive prenatal diagnosis of maternally inherited mutations has not been possible. Here we describe a digital relative mutation dosage (RMD) approach that determines if the dosages of the mutant and wild-type alleles of a disease-causing gene are balanced or unbalanced in maternal plasma. When applied to the testing of women heterozygous for the CD41/42 (-CTTT) and hemoglobin E mutations on HBB, digital RMD allows the fetal genotype to be deduced. The diagnostic performance of digital RMD is dependent on interplay between the fractional fetal DNA concentration and number of DNA molecules in maternal plasma. To achieve fetal genotype diagnosis at lower volumes of maternal plasma, fetal DNA enrichment is desired. We thus developed a digital nucleic acid size selection (NASS) strategy that effectively enriches the fetal DNA without additional plasma sampling or experimental time. We show that digital NASS can work in concert with digital RMD to increase the proportion of cases with classifiable fetal genotypes and to bring noninvasive prenatal diagnosis of monogenic diseases closer to reality.digital PCR ͉ fetal DNA ͉ mass spectrometry ͉ microfluidics ͉ thalassemia

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...

At midpregnancy, sonographic markers can effectively differentiate normal pregnancies from those with fetal hemoglobin Bart disease. Among couples at risk with no sonographic markers, the risk of having an affected child is nearly eliminated. The most sensitive marker was the cardiothoracic ratio, followed by placental thickness.