Novel Real-Time Quantitative PCR Test for Trisomy 21

Abstract

The detection of gross chromosomal abnormalities is a major focus of invasive prenatal diagnosis testing, of which the most common cytogenetic anomaly in live births is trisomy 21 (Down syndrome). Classically these examinations are lengthy procedures relying on karyotypic analysis of cultured amniocytes or chorionic mesenchyme. More rapid alternatives, such as fluorescence in situ hybridization and quantitative fluorescent PCR on uncultured cells, are time- and labor-intensive. Here we describe a novel real-time PCR (1)(2) assay for the detection of trisomy 21 that is readily amenable to automation and high-throughput screening. Real-time PCR can determine subtle alterations in gene dosage, such as hetero- or homozygosity of the RhD locus (3) and protooncogene amplification in breast cancer patients (4). We examined whether a real-time PCR assay could determine chromosomal ploidy, in particular for the prenatal detection of Down syndrome (trisomy 21). We examined the coamplification of a genetic locus (amyloid gene) in the Down’s region of chromosome 21 and a control locus [glyceraldehyde 3-phosphate dehydrogenase (GAPDH)] on chromosome 12. The amyloid gene locus on chromosome 21 was chosen because aberrant expression of this protein has been implicated in the physiologic lesions associated with Down syndrome (5). Furthermore, use of this locus should enable the detection of unbalanced Robertsonian translocations involving the Down’s region of chromosome 21, which occur in ∼4% of Down syndrome cases (6). No such cases were available during in this pilot study. We used a …