Rapid detection of common autosomal aneuploidies by quantitative fluorescent PCR on uncultured amniocytes

Abstract

Prenatal diagnosis of chromosomal abnormalities by cytogenetic analysis is time consuming, expensive, and requires highly qualified technicians. Rapid diagnosis of aneuploidies followed by reassurance for women with normal results can be performed by molecular analysis of uncultured foetal cells in less than 24 h. Today, all molecular techniques developed for a fast diagnosis of aneuploidies rely on the semiquantification of fluorescent PCR products from short tandem repeat (STR) polymorphic markers. Our objective was to test a chromosome quantification method based on the analysis of fluorescent PCR products derived from non-polymorphic target genes. An easy to set up co-amplification of portions of DSCR1 (Down Syndrome Critical Region 1), DCC (Deleted in Colorectal Carcinoma), and RB1 (Retinoblastoma 1) allowed the molecular detection of aneuploidies for chromosomes 21, 18 and 13 respectively. Quantitative analysis was performed in a blind prospective study of 400 amniotic fluids. Four samples (1%) could not be analysed by PCR probably because of a low concentration of foetal DNA. Follow up karyotype analysis was done on all samples and molecular results were in agreement with the cytogenetic data with no false -positive or false -negative results. Our gene based fluorescent PCR approach is an alternative molecular method for a rapid and reliable detection of aneuploidies which can be helpful for the clinical management of high-risk pregnancies.

Strategies for the rapid prenatal diagnosis of chromosome aneuploidy

“…The second quantitative PCR approach uses different primer pairs to multiplex three nonparalogous sequences (one each from chromosomes 13, 18 and 21). 22 Comparison of the relative amplification of these sequences was used to determine genomic copy number; 400 uncultured amniotic fluid samples were tested and all results were in agreement with the results of the karyotype analysis. A more automated approach is the use of real-time PCR to assess the relative amounts of amplified nonparalogous sequences from different chromosomes.…”

“…Furthermore, the real-time PCR strategy does not require a separate analysis step, and therefore reduces the risk of contamination and sample mix-up. However, these quantitative PCR approaches have some limitations: while the QF-PCR approach described in this paper exploits a 2:1 diallelic ratio and/or a qualitative triallelic result, the two other PCR approaches require a more subtle 3:2 dosage ratio to identify trisomy and for this reason may be less sensitive for the detection of trisomy mosaicism; triploidy and MCC are not detectable if the fetus is female (and in these studies sex chromosome copy number was not investigated); comparison of sequences amplified with different primer sets in the nonparalogous approach is likely to make it more susceptible to variation in sample quality, although one reported failure rate 22 was just 1%…”

High-Resolution Copy-Number Variation Map Reflects Human Olfactory Receptor Diversity and Evolution

“…The qPCR results were normalized using qPCR results of the regulator of calcineurin 1 (RCAN1) gene (previously Down Syndrome Critical Region 1 gene (DSCR1)), presumed not to vary in copy number in normal individuals[24],[25]. For comparisons between microarray and qPCR results we performed a further normalization by log scale subtraction of the value for the designated reference sample NA19154.…”

X-linked adrenoleukodystrophy (X-ALD): clinical presentation and guidelines for diagnosis, follow-up and management

“…ABCD1 mutational analysis can be performed either on a fresh chorionic villus sample at 11–13 weeks of pregnancy or on amniotic cells obtained from amniotic fluid after centrifugation at 15–18 weeks of gestation [80]. In some countries, pre-implantation genetic diagnosis is available.…”