Digital fetal aneuploidy diagnosis by next-generation sequencing.

Since the identification of fetal lymphocytes in maternal blood in 1969, investigators have endeavored to develop genetics-based noninvasive prenatal diagnostics (NIPD) (1 ). A robust noninvasive approach would augment or potentially supplant amniocentesis and chorionic villus sampling, which, although gold standards, carry a risk of fetal loss. Despite considerable efforts, the use of fetal cells for NIPD has never reached clinical implementation because of their paucity (on the order of a few cells per milliliter of maternal blood) and concerns of fetal cell persistence in the maternal circulation between pregnancies (2 ). A new avenue was opened in 1997 by the discovery of circulating cell-free fetal DNA in maternal blood (3 ). Cell-free fetal DNA constitutes between 5% and 10% of the total DNA in maternal plasma and increases during gestation. It rapidly clears from the circulation postpartum, a feature that enhances its attractiveness as a per-pregnancy– specific analyte. Several clinical applications based on the analysis of cell-free fetal DNA have been developed. These assays include determining fetal Rh D status in Rh D-negative women (4 ), sex in sex-linked disorders (3, 5 ), and detection of paternally inherited autosomal recessive and dominant mutations (6 ). In the context of these successes, however, there remains the outstanding challenge of the use of cell-free fetal DNA for the detection of chromosomal aneuploidy, in particular trisomies 21, 18, and 13. A cell-free fetal DNA– based approach for chromosomal aneuploidy must overcome several technical hurdles. By virtue of being cell free, fetal DNA in the maternal plasma cannot be easily analyzed by traditional visualization methods such as fluorescence in situ hybridization. The high “background” of maternal DNA (cell free or from residual cells) further complicates analysis by diluting the fetal genetic information. To address these issues, investigators have worked to identify fetal-specific molecular markers. The origin of circulating cell-free fetal DNA is primarily the placenta, whereas maternal cell-free DNA is derived from maternal leukocytes (7 ). By studying differences in genomic DNA methylation between the placenta and paired maternal leukocytes, investigators have characterized placenta-specific epigenetic markers (8 ). The finding of circulating cell-free placenta-derived mRNA allowed the identification of placenta-specific mRNA production (9 ). These findings have been the basis for the development of techniques for determining fetal chromosomal dosage that quantify the allelic ratio of single-nucleotide polymorphisms present in circulating fetal and maternal RNA or DNA. Although promising, these approaches depend on allelic heterozygosity between the fetus and mother for the analyzed single-nucleotide polymorphisms. Multiple markers are therefore needed for genetically diverse populations. To overcome this limitation, investigators have pursued polymorphism-independent methods, the first of which was digital PCR (10, 11 ). In digital PCR, individual fetal and maternal circulating cell-free DNA fragments are amplified in wells or microfluidics compartments under limiting-dilution conditions. By amplifying a target locus, on chromosome 21 for example, and quantitatively comparing its amplification with a locus on a reference chromosome, one should be able to measure an imbalance in chromosome 21 dosage. Specifically, the total number of chromosome 21 amplifications (representing maternal plus fetal contributions) divided by the number of reference chromosome amplifications should yield a ratio indicating an overor underrepresentation of chromosome 21. Although the digital PCR approach is conceptually solid, the low percentage of cell-free fetal DNA in the maternal plasma sample requires the performance of thousands of PCRs to generate a ratio with statistical confidence. Ongoing investigations to enhance this method include the use of multiple target amplifications and enrichment of cell-free fetal DNA. In the setting of the above accomplishments, nextgeneration sequencing (NGS) has entered the picture. NGS comprises several recently developed technologies that share the fundamental approach in which clonally amplified DNA templates (or, most recently, single DNA molecules) are sequenced in a massively parallel fashion within a flow cell (12, 13 ). The chem1 Department of Pathology, University of Utah, Salt Lake City, UT; 2 ARUP Laboratories Institute for Experimental and Clinical Pathology, Salt Lake City, UT. * Address correspondence to this author at: ARUP Laboratories, 500 Chipeta Way, Salt Lake City, Utah 84108. Received November 25, 2009; accepted December 14, 2009. Previously published online at DOI: 10.1373/clinchem.2009.141267 3 Nonstandard abbreviations: NIPD, noninvasive prenatal diagnostics; NGS, nextgeneration sequencing. Clinical Chemistry 56:3 336–338 (2010) Editorials