Rapid and simple prenatal DNA diagnosis of Down's syndrome

doi:10.1016/S0140-6736(97)11090-X

The Lancet

Volume 352, Issue 9121, 4 July 1998, Pages 9-12

https://doi.org/10.1016/S0140-6736(97)11090-X Get rights and content

Summary

Background

Prenatal diagnosis of chromosomal abnormality requires cytogenetic analysis of amniotic fetal cells. The necessary culture time delays diagnosis, is expensive, and requires substantial scientific expertise. In a masked prospective study, we investigated the feasibility of PCR amplification of chromosome 21 markers for the prenatal diagnosis of Down's syndrome.

Methods

The study population consisted of 2167 pregnant women, undergoing amniocentesis for prenatal diagnosis. In this cohort at least 1.5 mL amniotic fluid was available surplus to the requirements for traditional diagnostic methods. DNA was extracted from the surplus amniotic fluid and amplified in fluorescence-based PCR reactions, with three small-tandem-repeat markers located on chromosome 21. The products of the reactions were analysed on a DNA sequencer to identify the presence of two or three copies of chromosome 21.

Findings

In 2083 (97·4%) of 2139 samples of amniotic fluid that were not macroscopically blood-stained, two DNA markers gave an informative and correct result, identifying 2053 fetuses as normal and 30 as having trisomy 21 Down's syndrome (as confirmed by cytogenetic analysis). An extra marker was informative in 32 of 41 other clear samples. Thus a total of 99·6% informative results was achieved with these three markers. Macroscopically bloodstained samples (28 [1·3%]) were unsuitable for DNA testing. They gave a typical but non-informative result. There were no false-positive or false-negative results.

Interpretation

The PCR-based DNA diagnostic test has great potential for improved prenatal diagnosis of Down's syndrome, with the advantage that results may be available within a day.

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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Citation Excerpt :
These markers are amplified with primers flanking them; either of the primer should be fluorescently labeled to aid in relative quantification of peak areas thereby predicting the dosage of chromosome indirectly (Adinolfi et al., 1997). The method has been validated by number of studies conducted in diverse population since it has introduced (Pertl et al., 1999a,b; Quaife et al., 2004; Verma et al., 1998). As a result, QF-PCR has become a choice for routine prenatal aneuploidy diagnosis in many of the western countries (Cirigliano et al., 2001; Schmidt et al., 2000).
Among the rapid aneuploidy detection methods, QF-PCR has now become an alternative tool for prenatal aneuploidy diagnosis concomitant with karyotyping. This method has been validated in many of the western clinics but in India no study was conducted to assess its utility as standalone procedure. The study was designed to answer the question whether QF-PCR can be implemented as a standalone diagnostic method for rapid aneuploidy diagnosis in our present clinical setup?
Study was conducted during March 2012 to August 2014 consisting of 270 prenatal samples that underwent for aneuploidy diagnosis. In addition to karyotyping, QF-PCR was also performed on these samples and the results were compared.
Of 270 samples screened, 262 samples showed euploid genome (125 normal male and 137 normal female). Eight samples were consistent with aneuploidy — four trisomy 21 male sample, 2 trisomy 21 female sample, 1 trisomy 18 samples and 1 Klinefelter sample. The specificity, sensitivity, positive prediction value and negative prediction values were 100% while false positive rate and false negative rate were 0%.
Outcome of this study strongly suggests that QF-PCR can be used as standalone procedure for targeted rapid aneuploidy diagnosis.
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Nous avons testé la capacité de ces deux méthodes moléculaires à détecter ces anomalies chromosomiques sur 400 liquides amniotiques ou villosités choriales, prélevés chez des femmes enceintes présentant un risque élevé d’aneuploïdies.
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ArticlesRapid and simple prenatal DNA diagnosis of Down's syndrome

Summary

Background

Methods

Findings

Interpretation

Introduction

Section snippets

Methods

Samples without blood-staining

Discussion

Lancet

Genomics

Am J Hum Genet

Am J Hum Genet

UK National External Quality Assessment Scheme. Report for Clinical Genetics 1996/97

Clin Cytogenet

Maternal cell contamination in uncultured amniotic fluid

Prenatal Diagn

Prenatal detection of trisomy 21 in uncultured amniocytes by fluorescence in situ hybridization: a prospective study

Prental Diagn

Rapid prenatal diagnosis of chromosomal aneuploidies by fluorescence in situ hybridization: clinical experience with 4500 specimens

Am J Hum Genet

Precise gene dosage determination by polymerase chain reaction; theory, methodology and statistical approach

Mol Cell Probes

Articles
Rapid and simple prenatal DNA diagnosis of Down's syndrome