Abstract
The aim of this study was use a simple and rapid procedure, called segmental duplication quantitative fluorescent polymerase chain reaction (SD-QF-PCR), for the prenatal diagnosis of fetal chromosomal aneuploidies. This method is based on the co-amplification of segmental duplications located on two different chromosomes using a single pair of fluorescent primers. The PCR products of different sizes were subsequently analyzed through capillary electrophoresis, and the aneuploidies were determined based on the relative dosage between the two chromosomes. Each primer set, containing five pairs of primers, was designed to simultaneously detect aneuploidies located on chromosomes 21, 18, 13, X and Y in a single reaction. We applied these two primer sets to DNA samples isolated from individuals with trisomy 21 (n = 36); trisomy 18 (n = 6); trisomy 13 (n = 4); 45, X (n = 5); 47, XXX (n = 3); 48, XXYY (n = 2); and unaffected controls (n = 40). We evaluated the performance of this method using the karyotyping results. A correct and unambiguous diagnosis with 100% sensitivity and 100% specificity, was achieved for clinical samples examined. Thus, the present study demonstrates that SD-QF-PCR is a robust, rapid and sensitive method for the diagnosis of common aneuploidies, and these analyses can be performed in less than 4 hours for a single sample, providing a competitive alternative for routine use.
Highlights
Chromosomal aneuploidies cause a number of syndromes attributable to chromosomal aberration, and these abnormalities are typically associated with severe mental retardation, multiple dysmorphic features, growth retardation, etc
Samples A total of 96 samples were collected in this study, of which 36 samples were obtained from patients with trisomy 21 (15 peripheral blood samples; 15 uncultured amniotic fluid samples and 6 cord blood samples); 2 samples were obtained from unbalanced translocation cases, including 46,XX,der(14;21)(q10; q10),+21; 4 cord blood samples were obtained from patients with trisomy 13; 6 samples from patients with trisomy 18 (3 uncultured amniotic fluid samples and 3 cord blood samples); 5 peripheral blood samples were obtained from 45, X patients; 3 peripheral blood samples were obtained from 47, XXX patients; and 2 peripheral blood samples were obtained from 48, XXYY patients
The karyotypes for all samples were previously validated through full karyotyping analysis, and these data were withheld from the technician who performed the SD-Quantitative fluorescence polymerase chain reaction (QF-PCR) assays
Summary
Chromosomal aneuploidies cause a number of syndromes attributable to chromosomal aberration, and these abnormalities are typically associated with severe mental retardation, multiple dysmorphic features, growth retardation, etc. The frequency of chromosomal aneuploidy is approximately 1 out of 160 live births in the human population [1] These diseases cannot be cured, and prenatal diagnosis is important to predict these syndromes. The prenatal diagnosis of chromosome aneuploidies is typically performed using G-band cytogenetic analysis with in vitro cultures of nucleated fetal cells retrieved through amniocentesis, chorionic biopsy or fetal blood sampling. This conventional cytogenetic technique detects a wide range of aberrations with high reliability and has become the gold standard for the detection of fetal chromosomal abnormalities since the 1970s [2]. This method is unable to detect submicroscopic duplications and deletions
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