Molecular Human Reproduction, Vol. 7, No. 10, 1001-1006,
October 2001
© 2001 European Society of Human Reproduction and Embryology
Reproductive genetics |
Clinical application of multiplex quantitative fluorescent polymerase chain reaction (QF-PCR) for the rapid prenatal detection of common chromosome aneuploidies
1 Departament de Genética Molecular, General Lab, Barcelona 08021, 2 Unitat de Biologia, Departament de Biologia Cel·lular, Fisiologia i Immunologia, Universitat Autònoma de Barcelona, E-08193 Bellaterra, Barcelona, 3 Departament de Genética, General Lab, Barcelona and 4 Secció de Genética, Hospital de Sant Joan de Déu, Esplugues de Llobregat, 08950 Barcelona, Spain 5 To whom correspondence should be addressed at: Genética Molecular, General Lab, c/Amigo 12, 08021 Barcelona, Spain. E-mail: v_cirigliano{at}hotmail.com
Abstract
The clinical application of quantitative fluorescent polymerase chain reaction (QF-PCR) for rapid prenatal detection of chromosome aneuploidies has been limited in most studies to the detection of autosomal trisomies. Recently it has been shown that a newly identified highly polymorphic marker, termed X22, which maps to the Xq/Yq pseudoautosomal region of the sex chromosomes, used together with the X-linked short tandem repeat (STR) HPRT, allows the accurate detection of gonosome aneuploidies. We have developed a rapid assay, which includes these STR markers together with a sequence of the amelogenin region of the sex chromosomes and selected highly polymorphic autosomal STR. Two more X chromosome markers, as yet not used in previous QF-PCR applications, were also included in the assay. The molecular test was then used in a clinical trial on 551 uncultured amniotic fluid samples, allowing the assessment of copy number for chromosomes X, Y and 21 in 100% of cases. In the course of this study, two fetuses with Turner's syndrome and one with Klinefelter's syndrome were identified along with 17 autosomal trisomies. The assay proved to be so efficient and reliable that in most aneuploidy cases, in which ultrasound findings were in agreement with the molecular result, therapeutical interventions were possible without waiting for the result of cytogenetic analysis.
aneuploidy/prenatal diagnosis/QF-PCR/STR
Introduction
Since 1993, it has been repeatedly shown that the diagnosis of selected chromosome aneuploidies can be rapidly and accurately achieved by quantitative fluorescent polymerase chain reaction (QF-PCR) amplification of genomic repeated sequences known as short tandem repeats (STR) (Mansfield, 1993
; Adinolfi et al., 1997). In the last few years, this approach has been successfully used in large experimental and clinical trials on hundreds of amniotic fluids and chorionic villous samples (Verma et al., 1998; Pertl et al., 1999a,Pertl et al., 1999b). Due to the early scarcity of highly polymorphic STR markers on the X and Y chromosomes, the great majority of studies focused on the prenatal detection of autosomal aneuploidies and fetal sexing (Adinolfi et al., 1997; Pertl et al., 1997). Lately, the prenatal detection of Turner's and Klinefelter's syndrome by QF-PCR has been reported (Shmidt et al., 2000).
Recently, it has been shown that the newly identified pentanucleotide repeat X22, which maps to the pseudoautosomal region (PAR 2) of the X and Y chromosomes, together with HPRT and a modified sequence of the amelogenin region, allows the accurate detection of sex chromosome copy number (Cirigliano et al., 1999; Chen et al., 2000a). The aim of this study was to include these sequences together with selected highly polymorphic autosomal STR, to develop a set of highly informative multiplex QF-PCR tests for the rapid prenatal detection of aneuploidies involving chromosomes X, Y, 13, 18 and 21. The assay, used in a clinical trial of 551 uncultured amniotic fluid samples, allowed the detection of all numerical variations of the chromosomes examined within 24 h from collection of the sample.
Materials and methods
Clear amniotic fluids (n = 551) were collected from women between 14 and 24 weeks of gestation, except in one case where amniocentesis was performed at 31 weeks. The most frequent indication for fetal sampling was advanced maternal age, followed by abnormal biochemical screening, ultrasound findings, and parental anxiety. Six more samples were macroscopically bloodstained but QF-PCR was attempted because of advanced gestational age (+21 weeks) with abnormal serum screening or ultrasound findings. After seeding culture dishes for cytogenetic analysis, 
0.5 ml of amniotic fluid (range 0.5–1.5 ml) was available for DNA extraction and rapid molecular diagnosis. In one case it was only possible to collect 1 ml of amniotic fluid; since such a small volume was not suitable for in-vitro culture, it was entirely used for QF-PCR analysis. DNA was prepared by incubating cell pellets with InstaGene Matrix (Bio-Rad Laboratories, CA, USA), with slight modifications to the manufacturer's protocol. Briefly, after centrifugation of the amniotic fluid, unwashed cell pellets were incubated with 80–200 µl of matrix at 70°C for 8 min and at 95°C for 4 min. This procedure allows DNA to be quickly extracted (about 15 min) without further manipulation of the sample tube.
Six multiplex QF-PCR assays were set up using selected highly polymorphic STR markers on chromosomes X, Y, 21, 18 and 13. The X22 pseudoautosomal pentanucleotide STR (Cirigliano et al., 1999) was included in the assay for the detection of sex chromosome anomalies, together with the X-linked HPRT and the modified amelogenin sequence (AMXY) which simultaneously allow the assessment of fetal sex and copy number of XY chromosomes (Sullivan et al., 1993; Cirigliano et al., 1999). In order to reduce the number of normal females homozygous for both X22 and HPRT (and thus uninformative), two as-yet-untested X-linked tetranucleotide STR (from the Cooperative Human Linkage Center database), DXS6803 and DXS6809, were included.
Highly polymorphic D21S1414 and D21S1411 (Adinolfi et al., 1997; Sherlock et al., 1998) were included in the assay to screen all samples for chromosome 21 aneuploidies. These STR map to the long arm of chromosome 21 (21q21–21q22.3) flanking the Down's syndrome critical region, thus allowing the detection of most partial trisomies due to unbalanced translocations (Valero et al., 1999). Two more STR, D21S1435 and D21S1412 (Adinolfi et al., 1997; Valero et al., 1999), were used to confirm trisomic samples as well as for cases of double homozygosity for the first two markers. D21S11, which amplifies the same locus as D21S1414 (but produces a 122 bp shorter amplicon), was also included in this multiplex assay.
D18S535 and D18S386 (Pertl et al., 1999a) were used to test all samples for chromosome 18 while D18S51 was added to test samples found to be double homozygous for the first two STR. D13S631 and D13S634 (Adinolfi et al., 1997; Sherlock et al., 1998) were used for chromosome 13; D13S258 (Pertl et al., 1999a) was also available for re-testing uninformative or trisomic samples.
All forward primers (TIB Molbiol, Berlin, Germany) were labelled with fluorescent molecules (Table I) which allow the assessment of size and amount of the corresponding PCR product. Primers producing amplicons of similar size were labelled with different fluorochromes allowing amplification to be carried out in the same reaction (Sherlock et al., 1998).
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As shown in Table II
, for the first multiplex PCR the following primers were used: Amelogenin (AMXY), D21S1414, D18S535 and D13S631. In the second reaction we co-amplified X22, HPRT, D21S1411, D18S386 and D13S634.
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Four further multiplex QF-PCR were set up in order to test samples for selected chromosomes; these included four STR and AMXY for chromosome X, four STR for chromosome 21 and three markers each for chromosomes 13 and 18 (Table I
).
Aneuploidy screening was performed by testing all amniotic fluid samples using the first two multiplex QF-PCR which include two STR for each chromosome and AMXY. All STR markers included in this first preliminary assay were selected because of their high level of heterozygosity (>75%) (Tables II and III). After being tested with the first two multiplex assays, samples diagnosed as aneuploid or uninformative for one chromosome were retested using the correspondent chromosome specific assay (Table I).
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QF-PCR amplification was standardized for all reactions in a final volume of 25 µl containing 4 µl of extracted DNA, 200 µmol/l dNTP, 4–35 pmol of each primer, 2 mmol/l MgCl2 in 1xTaq buffer and 1 U of Taq polymerase (Promega, Madison, WI, USA). Sufficient PCR mastermix was prepared in a positively pressurized room under a class 2 hood for at least 20 samples and aliquoted in 0.2 ml PCR tubes to be frozen until needed. This approach allowed the QF-PCR set up to be carried out in less than 30 min, including DNA extraction, and allowed the test to be performed in exactly the same PCR conditions for large batches of samples. After denaturation for 2 min at 95°C, hot start PCR was performed on a GeneAmp 9700 (PE Biosystems, Foster City, CA, USA) for 28 repeating cycles at 94°C for 35 s, 58°C for 35 s and 72°C for 40 s, with final extension for 15 min at 72°C. QF-PCR products (1 µl) were collected in 12 µl formamide and 0.3 µl size standard (500 TAMRA; PE Biosystems) before loading on an ABI 310 genetic analyser (PE Biosystems). After denaturation, capillary electrophoresis was carried out in denaturing conditions using POP4 polymer (PE Biosystems). Analysis of the results and calculations of peak areas were performed using GeneScan software 3.1 (PE Biosystems) as previously described (Pertl et al., 1996
; Adinolfi et al., 1997). Results
All 551 clear amniotic fluid samples were successfully amplified and the QF-PCR result was made available to the genetic counsellor within 24 h.
Fetal sexing was successfully achieved in all cases by amplification of the X/Y homologous region amelogenin. Male fetuses (n = 270) were identified by the detection of the Y-derived sequence together with the X-specific product. In all cases the ratio between these two fluorescent peaks also allowed the assessment of sex chromosome copy number. For the five chromosomes examined, 527 out of 551 samples gave a normal QF-PCR result and 92% of these samples (n = 488) gave a clear heterozygous pattern for at least one STR per chromosome following the first two multiplex PCR (Table II). The ratio between the two corresponding peaks was in all cases very close to 1:1 (maximum range 0.7 to 1.3:1). Due to the high polymorphism of the markers employed for the first two multiplex PCR, only 43 (8%) normal samples were found to be double homozygous for both chromosome-specific STR (Table III). These amniotic fluids had to be retested using the corresponding chromosome-specific multiplex assay in order to include one or more extra STR (Table I). Following the third PCR, all 551 samples were informative for copy number of chromosomes X, Y and 21, 99.8% of samples (n = 550) were also informative for chromosome 18 and 99.4% of samples (n = 548) were informative for chromosome 13 (Table III). The inclusion of the modified AMXY sequence together with the X22 STR greatly reduced the number of uninformative samples for sex chromosome copy number. In the course of this study, 92% of normal males were found to be heterozygous for the pseudoautosomal X22. In the remaining homozygous samples, the chromosome X and Y copy numbers could still be reliably diagnosed using the AMXY peak ratio alone.
Twenty samples with different aneuploidies were correctly identified; they included trisomy 21 (n = 7), trisomy 18 (n = 6), trisomy 13 (n = 3), 45, X (n = 2), 47, XXY (n = 1) and 69, XXX (n = 1). Cytogenetic analysis always confirmed the molecular results and additionally revealed one case of trisomy 22, that the rapid test was not designed to detect, and one case of mosaicism for an Xq26 deletion in only 12% of cells; too low to be identified by the QF-PCR assay (Cirigliano et al., 1999). The diagnosis of trisomy for one chromosome was given for samples showing characteristic triallelic patterns with a ratio between the three peaks of fluorescent activity close to 1:1:1 or diallelic with a ratio very close to 2:1 (maximum range 1.8 to 2.3:1) (Figures 1 and 2). In the one case of triploidy, triallelic and diallelic trisomic patterns were observed for all markers.
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The diagnosis of Turner's syndrome was based on the assumption that, due to the high polymorphism of the pseudoautosomal X22 and the three X-chromosome STR used, samples showing only one peak for all markers, in absence of a Y-specific product of amelogenin, were far more likely to result from an X monosomy than from homozygosity (Figure 2
). By combining the heterozygosity of these sequences, the probability for a normal female to be homozygous for all markers, and thus indistinguishable from Turner's syndrome, is very low (about 0.4%). This approach allowed the prenatal detection of two cases of Turner's syndrome.
One case of Klinefelter's syndrome was correctly identified using the AMXY marker because the X-specific product was present in a double dose compared to the Y (Figure 1). In this case, the trisomic triallelic pattern of the pseudoautosomal X22 further confirmed the presence of three sex chromosomes and HPRT of two X chromosomes.
Eighteen out of 20 aneuploid samples were readily detected with the first two multiplex PCR without false negative results. The remaining two were both amniotic fluids from fetuses with Turner's syndrome and were correctly identified following the sex chromosome specific multiplex PCR. All aneuploid samples were re-tested, to confirm the diagnosis, using the corresponding chromosome-specific QF-PCR assay (Table I and Figure 2). In all 20 aneuploidy cases, at least two STR were informative and, in 17 cases, termination of pregnancy was performed without waiting for the fetal karyotype but after further confirmation of the diagnosis by fetal ultrasound examination. Only in one case of trisomy 21, with apparently normal fetal ultrasound, was no action was taken until completion of cytogenetic analysis. The two remaining cases were both cases of trisomy 13; the first one, from a 31 week gestation, was spontaneously delivered a few days after amniocentesis; the second one was an amniotic fluid sample from a 20 weeks exitus.
In this trial, we could also confirm the great clinical utility of QF-PCR amplification of microsatellites in three cases of twin gestation. In the first one, the molecular result was compatible with Turner's syndrome for both fetuses that also had the same STR profile for all markers. This result was eventually compatible with a monozygotic twin pregnancy, but not concordant with ultrasound findings, suggesting Turner's syndrome in only one of the fetuses. The other possibility was that the same amniotic cavity had inadvertly been sampled twice. Amniocentesis was repeated only for the fetus with normal ultrasound and the QF-PCR result, available the same day, showed a normal male, thus confirming a dizygotic twin pregnancy with a male fetus and a Turner's fetus. Another twin pregnancy corresponded again to apparently monozygotic twins (normal females), but in this case, the QF-PCR results were concordant with ultrasound examination of chorionicity. Finally in the third case we could confirm the correct sampling of both amniotic cavities even in the presence of two normal female fetuses, because of their different STR profiles.
Rapid molecular detection of aneuploidies can be hampered by the presence of contaminating maternal cells in the amniotic sample. For this reason, in this trial we routinely excluded dark bloodstained amniotic fluids. However, in six cases of heavily bloodstained amniotic samples, we were asked to attempt the QF-PCR test because of advanced gestational age (>21 weeks) with abnormal biochemical screening or ultrasound findings. One case resulted in the diagnosis of a normal male fetus and, surprisingly, without any trace of maternal cell contamination. In two cases in which QF-PCR showed apparently normal female fetuses, maternal blood was made available and tested with the same STR used on amniotic fluid. This allowed the assessment and quantification of maternal cell contamination. In one case, we could diagnose a normal female fetus with low level maternal DNA contamination; the second one was the opposite situation, a very low amount of fetal DNA and a majority of maternal DNA, therefore uninformative for fetal chromosome copy number. Finally, the remaining three cases produced the characteristic pattern of a fetal maternal mixed sample and no result could be obtained other than fetal sex.
Discussion
We have developed a new, highly informative combination of STR markers for the rapid prenatal detection of non-mosaic aneuploidies involving chromosomes X, Y, 21, 13 and 18. Multiplex QF-PCR amplification of selected microsatellites was used as a preliminary test on 551 clear amniotic fluids before completion of cytogenetic analysis. With the STR selected for the assay, in most cases (92%), the result for the five chromosomes was achieved following the first two multiplex QF-PCR, thus in a very short period of time (even less than 5 h in urgent cases). In all aneuploid samples, the next chromosome-specific PCR allowed the diagnosis to be confirmed with at least two different markers. The STR included in the assay allowed the assessment of copy number for chromosomes X, Y and 21 in 100% of samples tested. Because of the lack of highly polymorphic sex chromosome STR, most previous QF-PCR investigations on prenatal samples were limited to the detection of autosomal trisomies and fetal sex. More recently, a newly identified pseudoautosomal pentanucleotide repeat, the X22, was shown to be highly polymorphic (12 alleles) and of diagnostic value for the detection of sex chromosome aneuploidies (Cirigliano et al., 1999). In the first trial, performed on samples previously diagnosed by conventional cytogenetic analysis, it allowed the assessment of sex chromosome copy number in most of the males and females tested, and it was therefore also possible to perform rapid diagnoses of Turner's and Klinefelter's syndromes (Cirigliano et al., 1999).
The inclusion of the X22 STR in two multiplex QF-PCR assays, together with two new X-linked markers (DXS6803 and DXS6809), HPRT and the modified AMXY sequence allowed, in this clinical trial, the rapid prenatal assessment of sex chromosome copy number in all 551 samples tested. Two fetuses with Turner's and one with Klinefelter's syndromes were also correctly identified.
In another recent clinical trial, it was also possible to detect sex chromosome aneuploidies in uncultured amniotic fluids by QF-PCR (Schmidt et al., 2000). The use of the modified sequence of amelogenin together with three X-Linked STR (SBMA, DXS8377, and DXS 1283E) allowed the authors to diagnose both Klinefelter's and Turner's syndromes. However, with the inclusion of the pseudoautosomal X22 together with AMXY in the present multiplex QF-PCR test, in most cases of normal male fetuses (heterozygous for this marker), we had two sequences confirming the same result, thus increasing the reliability of the diagnosis. Furthermore, it is always recommended to confirm a trisomic diallelic pattern with at least another marker, and the use of X22 also allows this confirmation in Klinefelter's (and XYY) samples that otherwise can only be detected with AMXY primers.
The molecular detection of gonosome mosaicism, however, is still hampered by the sensitivity of QF-PCR to detect low percentages of cell lines with X monosomy. In the first trial performed on blood samples (Cirigliano et al., 1999), it was shown that mosaics 46,XX (90%)/45,X (10%) are not detectable by the assay. This has already been confirmed on prenatal samples (Schmidt et al., 2000) and further shown here. It was possible instead to identify the presence of different proportions of XY cell lines by detecting Y-specific products of the amelogenin. In more complex mosaic situations, the presence of extra X22 and HPRT alleles with skewed ratios was not compatible with normal chromosome complements so that mosaicism could eventually only be suspected (Cirigliano et al., 1999).
In this clinical trial, in only four out of 551 samples were the three autosomal STR used homozygous (i.e. uninformative): these were three cases involving chromosome 13 and one involving chromosome 18, all of which were later shown to be normal by cytogenetic analysis. For chromosomes 13 and 18, we only used three STR; the eventual addition of a fourth marker will further reduce the number of normal uninformative samples, as achieved for chromosomes X, Y and 21. However, as shown here, it is such a rare condition for a normal disomic to be homozygous for three highly polymorphic STR, that the probability of a trisomic sample showing the same pattern (and not being detected by the present QF-PCR assay) will be even lower.
Clear samples showing the presence of only a few blood cells after centrifugation were always amplified without any evidence of maternal contamination in the resulting products. QF-PCR amplification of highly polymorphic STR on a sample suspected to be heavily contaminated with maternal blood cells is expected to produce a characteristic pattern with extra alleles or skewed ratios between peaks for all chromosomes. These are not usually compatible with a normal or with a trisomic result, so there is no risk of misdiagnosis. Another possibility in dark bloodstained fluids is the presence of a low level of maternal cell contamination producing extra peaks of very little fluorescent activity that do not significantly alter the ratio between fetal STR peaks. In these cases, and in the presence of a male fetus, diagnosis can be performed without great difficulty, but if the fetus is female it is necessary to confirm the fetal origin of the predominant cell population in the sample by testing maternal blood with the same markers. Although we suggest that the test on bloodstained amniotic samples should not be performed, in six cases we were asked to attempt a rapid diagnosis, and in two such cases (one male and one female fetus) results for all five chromosomes could be obtained.
In this clinical study, the QF-PCR result was always made available to the referring physician within 24 h from collection of the sample. In most cases of aneuploidy, it was a confirmation of previous abnormal fetal ultrasound findings and, in 17 out of 20 cases, legal therapeutic interventions were made possible on the basis of QF-PCR. In the majority of cases, where no abnormalities were found, this test was of great help to the parents to reduce the anxiety caused by an abnormal result of previous non-invasive (serum or ultrasound) screening.
If compared with FISH on uncultured amniocytes, QF-PCR as an adjunct to standard karyotyping has the same limitation of detecting only selected chromosome aneuploidies, but has several technical advantages that increase its clinical utility.
The PCR amplification with fluorescent primers is far more sensitive than hybridization and the assay is also reproducible and reliable if performed on minute amounts of DNA (Sherlock et al., 1998). As shown here, a small aliquot of amniotic fluid (0.5 ml), is usually sufficient to perform the test. The low volume needed for the assay also makes QF-PCR applicable in cases where rapid diagnosis is not previously planned, such as slow-growing or contaminated cytogenetic cultures. The efficiency is not influenced by the gestational age, and in this study we did not observe the high rate of uninformative samples that has been reported for FISH analyses (Bryndorf et al., 1997). Apparently clear samples showing only a little contaminating blood after centrifugation, which could hamper FISH especially with female fetuses, have all been successfully amplified and diagnosed. In dark bloodstained amniotic samples, maternal cell contamination was always readily detected as a peculiar STR pattern, without any risk of misdiagnosis. The analysis of the STR profile also allowed the assessment of zygosity in three cases of twin gestations as already reported (Chen et al., 2000b). Furthermore, the automation of the DNA scanner allows a high throughput of samples (Verma et al., 1998); in this trial we standardized all laboratory procedures and a single operator could easily handle up to 15 samples at the same time, with the result always available the next working day.
QF-PCR is a valid alternative for rapid aneuploidy screening, since all the above-mentioned advantages, together with the low cost of the procedure, make possible its application in low risk pregnancies as a preliminary tool to reduce parental anxiety before completion of cytogenetic analysis.
Acknowledgements
We would like to thank Prof. M.Adinolfi and Dr J.Sherlock for useful discussions and suggestions throughout the course of the study.
References
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Submitted on March 15, 2001; accepted on July 18, 2001.
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