Molecular Human Reproduction, Vol. 5, No. 12, 1176-1179,
December 1999
© 1999 European Society of Human Reproduction and Embryology
Diagnosing genetic disease |
Rapid prenatal diagnosis of aneuploidy by quantitative fluorescent PCR on fetal samples from mothers at high risk for chromosome disorders
1 Department of Obstetrics and Gynecology, University of Graz, Auenbruggerplatz 14, A-8036 Graz, 2 Department of Human Genetics, University of Graz, Austria, and 3 Department of Obstetrics and Gynaecology and The Galton Laboratory, University College, London, UK
Abstract
We report the results of a prospective study using quantitative fluorescent polymerase chain reaction (QF-PCR) and small tandem repeat markers (STR) for the rapid prenatal detection of aneuploidies in a group of pregnant women at increased risk of having fetuses with numerical chromosome disorders. Amniotic fluid samples (n = 52) were collected from mothers undergoing prenatal invasive testing for fetal abnormalities on ultrasonographic examination or abnormal maternal serum aneuploidy screening results. All samples were tested by cytogenetic analysis, but rapid diagnoses of aneuploidies were offered and performed using QF-PCR analysis with several STRs specific for chromosomes 21, 18, 13 and X. All cases with numerical chromosome aberrations involving chromosomes 21, 18 and 13 (n = 8) were correctly diagnosed. Three gonosomal aneuplodies (one 47,XXY and two 45,X) were not detected because they were uninformative for the X markers. Another sample with a deletion (46,XX,7q-), that the present assay was not designed to detect, was not identified. One sample was heavily contaminated with maternal blood and the results of the QF-PCR assays were uninformative. The remaining samples from normal fetuses provided QF-PCR patterns disomic for chromosomes 21, 18, 13 and X. Our study demonstrates that QF-PCR is a rapid method for the detection of common numerical chromosome disorders and it may play an important role in prenatal diagnosis for women at high risk for fetal aneuploidy.
aneuploidies/prenatal diagnosis/quantitative fluorescent PCR
Introduction
During the past decade, there has been a considerable progress in further refining non-invasive methods for the prenatal detection of fetal diseases. Biochemical and ultrasound tests have become standard procedures for screening for fetal chromosomal abnormalities (Norton, 1994
). Both approaches imply that a high proportion of tested mothers are told that their fetuses may have a major chromosome disorder and that an invasive procedure is required to confirm the diagnosis.
The interval between the collection of the fetal sample and the time of completing the cytogenetic analysis of the fetal karyotype is a period of great anxiety for the parents. Various approaches have been tried to accelerate prenatal diagnosis of aneuploidy, including chorionic villus sampling (CVS), early amniocentesis and/or analysis of uncultured cells by fluorescence in-situ hybridization (FISH) (Klinger et al., 1992; Eiben et al., 1998). However, some of these procedures are associated with an increased risk of miscarriage or technical difficulties which currently limit their wide clinical application.
Recently, a quantitative fluorescent polymerase chain reaction assay (QF-PCR) has been developed for the rapid prenatal detection of sex and aneuploidies involving chromosomes 21, 18 and 13 using small tandem repeat (STR) markers, specific for each type of chromosome (Mansfield, 1993; Pertl et al., 1994, 1997, 1999; Toth et al., 1998, Verma et al., 1998). This technique allows the detection of the major chromosomal abnormalities within 24 h after amniocentesis, CVS or fetal blood sampling (FBS).
In this study, we evaluated the possibility of using QF-PCR to confirm the presence of chromosome aneuploidies in cases in which ultrasound examinations or biochemical tests had suggested fetal disorders and a decision had to be made about the outcome of the pregnancy. QF-PCR and STR markers specific for chromosomes 21, 18, 13 and X were used as previously described (Pertl et al., 1997). Furthermore, we introduced three additional markers specific for chromosomes 18, 13 and X to allow diagnosis in samples homozygous for specific STRs, thus reducing the rate of uninformative results.
Materials and methods
Over a 3 year period QF-PCR was applied to amniotic fluid samples (n = 52) as an adjunct to conventional cytogenetic analysis. Fetal chromosome abnormalities were suspected on the basis of: (i) fetal structural abnormalities detected by prenatal ultrasound examination (n = 40); (ii) intrauterine growth retardation (n = 9); and (iii) abnormal maternal serum aneuploidy screen results (n = 3).
All mothers were informed about the aim of applying QF-PCR for rapid prenatal diagnosis of aneuploidies. However, in this study no immediate action was taken about the outcome of the pregnancies based only on the results of the QF-PCR tests. All diagnoses assessed by the QF-PCR tests were confirmed by the results obtained using cytogenetic tests before a final decision about possible intervention was made. Thus, the present work should be seen as a pilot study performed in order to evaluate the clinical value of QF-PCR and STRs for the rapid prenatal diagnosis of aneuploidies in a group of women at high risk of having fetuses with numerical chromosome aberrations.
A multiplex QF-PCR assay was applied, using STRs specific for chromosomes 21, 18 and 13 as previously described (Pertl et al., 1997). DNA was extracted with a standard phenol–chloroform extraction procedure (Sambrook et al., 1989) from uncultured amniotic fluid (1–5 ml). The primers used for the different STR markers and for sexing are listed in Table I.
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PCR amplification was performed in a total volume of 25 µl containing genomic DNA, 200 µmol/l dNTPs, 5–20 pmoles of each primer, 1x Taq polymerase buffer (3 mmol/l MgCl2) and 1.5 IU Taq polymerase (both Promega, USA). After denaturation at 94°C for 5 min, hot start PCR was carried out for 22 cycles at 94°C for 48 s, 60°C for 48 s and 72°C for 1 min. Final extension was for 5 min at 72°C.
Two separate multiplex PCR assays were designed using the following markers: first set: D21S1414, AMXY, MBP, D13S631 and D13S634; second set: XHPRT, D18S535, D21S1412, D21S1411 and D21S11. Additional markers (D13S258, D18S386, DXS337) were used in case of homozygosity of all markers specific for one chromosome. The PCR products (3 µl) were mixed with 2.6 µl of loading buffer and 0.4 µl of Genescan-500 Rox containing the reference molecular size standard. Electrophoretic analysis was performed using a 6% denaturing polyacrylamide gel and employing the model 373A DNA sequencer (Applied Biosystems Inc, USA). The amplification products were analysed and their relative fluorescent intensities calculated using Genescan 672 software (Applied Biosystems Inc) as previously described (Pertl et al., 1997; Verma et al., 1998) (Figures 1 and 2).
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Results
Amniotic fluid samples were analysed by QF-PCR before the cytogenetic tests were completed. The results of the QF-PCR analysis were then compared with those obtained by conventional cytogenetic tests. The mean gestational age at amniocentesis was 24.3 weeks (range 14–38 weeks).
Cytogenetic analysis showed that 40 samples had normal and 12 samples abnormal chromosome complements (two cases of trisomy 21, three cases of trisomy 18, three cases of triploidy, one case of 47,XXY, two cases of 45,X and one case of 46,XX,7q-) (Table II).
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QF-PCR successfully identified all numerical aberrations involving chromosomes 21, 18 and 13; all autosomal aneuploidies (two cases of trisomy 21, three cases of trisomy 18, 3 cases of triploidy) were found in the abnormal ultrasonography group. Three chromosomal aneuploidies (47,XXY; two cases of 45,X) were not detected by QF-PCR, because the markers specific for chromosome X showed a homozygous STR pattern and, therefore, were uninformative. Another sample had an abnormal karyotype (46,XX,7q-) that the QF-PCR assay was not designed to identify.
Out of 40 karyotypically normal samples, 39 were correctly identified as disomic for all chromosomes tested by QF-PCR. One amniotic fluid sample was visibly contaminated with maternal cells. This sample was tested by QF-PCR according to the standard protocol, together with the corresponding maternal blood sample. By comparing the STR patterns of the maternal and the amniotic fluid sample, maternal cell contamination was clearly detectable since additional peaks for all informative markers in the fetal sample had exactly the same sizes of those present in the maternal sample.
Sexing by QF-PCR was performed by amplification of the amylogenin-like DNA sequence AMXY, expressed on chromosomes X and Y. All results were found to be in agreement with cytogenetic tests.
Discussion
The present study demonstrates the potential clinical value of using QF-PCR for the rapid prenatal detection of aneuploidies in samples retrieved from mothers at increased risk of having fetuses with numerical chromosome abnormalities. Our data demonstrate that QF-PCR analysis allows accurate prenatal identification of selected numerical abnormalities within 24 h after sampling.
One case of Klinefelter syndrome and two cases of Turner syndrome were not detected by QF-PCR assays using the X chromosome markers XHPRT and DXS337 which have low polymorphism and produce a high rate of homozygous and, hence, uninformative results (Pertl et al., 1999). However, recent investigations have shown that it is possible to perform prenatal diagnosis of numerical disorders of the sex chromosomes by using two chromosome X markers (X22 and HPRT) (Cirigliano et al., 1999).
One visibly blood-stained amniotic fluid sample showed additional peaks and distorted ratios of the fluorescent PCR peaks. The high sensitivity of the QF-PCR assay allows the detection of contaminating maternal cells, even if present in a very low number. When samples suspected of being contaminated were tested simultaneously with the corresponding maternal blood specimens, contamination could be detected by comparing the fetal and maternal STR patterns. The analysis of the STR profiles of the fetal and the corresponding maternal samples provides a clear and accurate evaluation of possible contamination because the extra fluorescent peaks in the fetal sample will have the same size of those present in the mother. However, in cases of clear amniotic fluid samples there is no need to determine the maternal STR profile.
With regard to the restriction that only major chromosome abnormalities can be detected by QF-PCR, there are obvious similarities to FISH on uncultured amniocytes. However, compared with FISH analysis of prenatal samples the PCR-based method is less expensive and has the major advantage that it can be automated (Verma et al., 1998). FISH analysis is a labour-intensive and time-consuming technique which potentially limits its application for a high throughput of samples. Using the QF-PCR assay a single operator can analyse about 20 samples per day, depending on the equipment used. Moreover, the relatively high percentage of uninformative results obtained by FISH analysis of amniotic fluid samples at late gestational age (Bryndorf et al., 1997; D'Alton et al., 1997) was not observed in our study using QF-PCR. Although the overall number of samples is low, QF-PCR analysis was informative in all samples (n = 12) at late gestational age (>28 weeks). The phenomenon of this rather high percentage of uninformative results obtained by FISH is not fully understood, it could be related to an insufficient cell number for FISH analysis or an increased degradation of the chromatin in the amniotic cells at late gestational age. Our results seem to indicate that QF-PCR might be informative in a higher percentage at late gestational age.
Rapid analysis of chromosomes 21, 18, 13, X and Y may be of great help in pregnancies at high risk when the woman is close to the legal limits of termination of pregnancy or in cases of late gestational age when a decision has to be made on further clinical management.
Currently, rapid karyotyping is accomplished by FBS and placental biopsy. However, FBS is associated with a fetal loss rate of near 2%; an even higher loss rate has been reported, when the indication for FBS is a structural anomaly detected on ultrasound (13%) (Antsaklis et al., 1998). Amniocentesis with QF-PCR analysis could, therefore, be a safer alternative for rapid aneuploidy detection.
The results of our study demonstrate that QF-PCR may be a useful adjunct to karyotype analysis for high risk situations. The major advantages of using the QF-PCR method for the prenatal diagnosis of selected numerical chromosome abnormalities are the rapidity of the test and its accuracy at all gestational ages, which allow the confirmation of clinically suspected aneuploidies.
Acknowledgments
FWF was supported by grant No. P10470-Med from the Austrian Science Foundation, Vienna, Austria.
Notes
4 To whom correspondence should be addressed 
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Submitted on June 30, 1999; accepted on September 17, 1999.
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