ArticlesRapid and simple prenatal DNA diagnosis of Down's syndrome
Introduction
Since the early 1970s, prenatal diagnosis of Down's syndrome by karyotyping has been possible, generally by use of cultured amniotic-fluid cells obtained by amniocentesis at around 16 weeks of gestation. Each year in the UK, for example, nearly 40 000 women undergo amniocentesis, the majority because of an increased risk of having a child with trisomy 21 Down's syndrome. Chromosome analysis and karyotyping for the identification of fetal trisomy 21 Down's syndrome can be done only during the metaphase stage of the cell cycle. The culturing of amniocytes requires around 10 mL fluid and takes an average of 15 days.1 The inevitable delay between amniocentesis and fetal chromosome diagnosis causes much parental anxiety. Other drawbacks of this technique include the risk of culture failure, which occurs in nearly 1% of cases, and also the possibility of misleading results due to overgrowth of maternal cells, estimated to occur in around 0·2% of cultures.2
In the late 1980s, the introduction of fluorescence in-situ hybridisation (FISH), a technique in which chromosomes are targeted with fluorescence-labelled DNA probes, specific for the region or chromosome of interest, enabled identification of trisomy 21 in interphase cell nuclei of uncultured amniocytes.3 In the USA this method has now been introduced into diagnostic service in several centres as an adjunct to traditional cytogenetics.4 Although the FISH technology is used throughout service laboratories in the UK for the diagnosis of various genetic disorders, it has not been generally applied for prenatal diagnosis of Down's syndrome.
An alternative approach for the detection of trisomy 21 involves PCR amplification of small-tandem-repeat (STR) markers located on chromosome 21, and analysis by fluorescence-based methods to identify the presence of an additional allele on the third copy of the chromosome. This DNA approach was first used for the diagnosis of X-chromosome aneuploidy,5 and has since been modified to include the identification of some other common trisomies.6, 7 Small pilot studies have assessed the clinical usefulness of this technique.8, 9 The main advantages of this approach for the prenatal diagnosis of trisomy 21 Down's syndrome, on DNA extracted from uncultured samples of amniotic fluid, are the small amount of fluid required and the speed and accuracy with which the test can be done. The technique is automated, with the potential for 36–96 samples to be processed simultaneously, and results are available within the same day. It is also objective, requiring a minimum of expertise for interpretation.
In a masked prospective study, we investigated the use of PCR amplification of STR markers on chromosome 21 for the diagnosis of trisomy 21 in a series of more than 2000 samples of amniotic fluid. We looked at diagnostic sensitivity and reliability, costs, and time taken to obtain a result.
Section snippets
Methods
A small volume of amniotic fluid, surplus to the requirements for cell culture for karyotyping and measurement of ä-fetoprotein, was obtained from each of 2167 women referred to the Regional Cytogenetics Laboratory at Birmingham Heartlands Hospital, UK. The referral criteria were maternal age 35 years and older, positive screening test for Down's syndrome by maternal serum variables, an abnormal fetal ultrasonographic scan, or an increased risk of chromosomal fetal abnormality such as a
Samples without blood-staining
2139 (98·7%) of the samples of amniotic fluid were clear, without any macroscopically detectable blood-cell contamination. With the two markers D21S11 and IFNAR, 2083 (97·4%) of these samples gave informative results. For 2053 cases, two peaks of roughly equal intensity (ratio about 1) were seen (figure, A). They were therefore predicted to be normal, which was confirmed cytogenetically. 30 samples were diagnosed as Down's syndrome, since they showed either three STR peaks of equal intensity (
Discussion
In this masked prospective study we have shown that PCR-based DNA identification of Down's syndrome is a rapid and reliable technique for prenatal diagnosis. The test is informative whenever the markers on the parental chromosomes differ in size, which is dependent on the natural variation in the population studied. The results were strikingly different for clear and blood-stained fluid samples. One advantage of this approach is that results are obtained within the same day, compared with about
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