Molecular Human Reproduction, Vol. 6, No. 9, 855-860,
September 2000
© 2000 European Society of Human Reproduction and Embryology
Genetic diagnosis |
Detection of aneuploidy in chromosomes X, Y, 13, 18 and 21 by QF–PCR in 662 selected pregnancies at risk
1 Institute of Immunology, Pathology, Molecular Biology and Human Genetics (IPM), Lademannbogen 61–63, 22339 Hamburg, and 2 Allgemeines Krankenhaus Barmbek, Dept. of Prenatal Diagnosis and Therapy, Rübenkamp 148, 22307 Hamburg, Germany
Abstract
A quantitative fluorescent–polymerase chain reaction (QF–PCR) test system with different short tandem repeat (STR) markers of the X chromosome (SBMA, DXS8377 and DXS1283E) together with the amelogenin locus (AMXY) was developed for the rapid detection of sex chromosome aneuploidies on uncultured amniotic fluids. The samples (n = 662) were also tested with STRs specific for chromosomes 13, 18 or 21, with two STRs used for each chromosome. In uninformative cases, an additional STR marker was applied. The QF–PCR data were compared with the results of conventional cytogenetics. One dark red stained specimen showed an artificial PCR pattern, probably due to maternal contamination. Six sex chromosome aberrations (four 45,X, one 47,XXY, one mosaic 47,XXY/46,XX) were identified as aneuploid by STRs specific for chromosome X and AMXY. One pregnancy with a mosaic 45,X/46,XX karyotype was not detected by the assay. In all, 12 cases with a numerical aberration involving either chromosome 18 or 21 or with a triploidy were correctly diagnosed by QF–PCR. No information was obtained in one fetal sample with a trisomy 18 due to an uncertain result for two of the three applied STRs specific for chromosome 18 and an uninformative third STR marker. Two samples with an unbalanced Robertsonian translocation could be identified by QF–PCR as trisomic for chromosomes 13 and 21 respectively. The results show an excellent agreement between QF–PCR and cytogenetics with regard to sex chromosome and autosomal aneuploidy detection in prenatal diagnosis.
amniocytes/prenatal diagnosis/quantitative fluorescent-polymerase chain reaction (QF-PCR)/sex chromosome aberrations/trisomy 13, 18 and 21
Introduction
Since 1992, fluorescence in-situ hybridization (FISH) on uncultured amniocytes of pregnancies at risk has been used for the rapid detection of numerical aberrations of chromosomes X, Y, 13, 18 and 21 (Klinger et al., 1992
; Ward et al., 1993; Bryndorf et al., 1997; Eiben et al., 1998, 1999; Jalal et al., 1998; Lewin et al., 2000; Thilaganathan et al., 2000). An alternate approach for the identification of selected aneuploidies is the use of fetal DNA amplified by quantitative fluorescent–polymerase chain reaction (QF–PCR) using short tandem repeats (STRs) (Mansfield, 1993; Pertl et al., 1996; Findlay et al., 1998; Toth et al., 1998; Verma et al., 1998). Recently, the first diagnostic application of this method was described in a systematic study (Verma et al., 1998). This group screened 2167 samples of uncultured amniotic fluid for the prenatal diagnosis of trisomy 21. Other authors (Pertl et al., 1999) demonstrated the detection of major autosomal aneuploidies by QF–PCR, analysing a large number of chorionic villus samples. So far, the low level of polymorphism of most X-specific DNA markers has hampered the use of QF–PCR for the detection of sex chromosome aberrations. Recently, a first investigation on peripheral blood samples of patients with sex chromosome aberrations previously diagnosed by cytogenetic analysis showed that it is possible to detect patients with a Turner syndrome or an XXY chromosome constitution using X22, HPRT and P39 as X-linked markers (Cirigliano et al., 1999). We tested three other STR markers for the X chromosome for the identification of sex chromosome aneuploidies in prenatal diagnosis. We focused our study on 662 amniotic fluid samples of pregnancies at risk, thereby studying the most relevant fetal sex chromosome and autosomal aneuploidies.
Materials and methods
Patients and cytogenetic analysis
The selected indications of prenatal diagnosis included advanced maternal age (>35 years, n = 341), abnormal fetal ultrasonographic signs with or without advanced maternal age (n = 134), positive test results after maternal blood biochemical screening methods (n = 75), a previous fetus or child with a chromosomal aberration (n = 15) or other indications (n = 97). These indications were parents' anxiety (n = 73), pregnancies complicated by a feto–fetale transfusion syndrome (n = 18), X-chromosomal defects (n = 3) and maternal virus infections (n = 3). At least 3–6 ml of amniotic fluids were used for cell cultivation and subsequent cytogenetic analysis according to standard techniques. A minimum of 11 metaphases was analysed.
Quantitative fluorescence–polymerase chain reaction (QF–PCR)
Genomic DNA was extracted from 1–2 ml of uncultured amniotic cells using a QIAamp blood kit (Qiagen, Germany). All samples were processed including visibly light or dark red stained amniotic fluids with potential maternal cell contamination. Visibly light or dark red samples were washed twice with aqua bidest. All DNA samples were coded and the analysis was undertaken without knowledge of the fetal karyotype. For sex determination the unique sequence in the first intron of the X/Y homologous gene amelogenin (AMXY) was amplified (Nagafuchi et al., 1992). For the detection of sex chromosome aberrations and selected autosomal trisomies two different STR markers were used per chromosome. In uninformative cases, a third STR marker was employed. The STR markers were selected from genome data because of their high heterozgosity rates and good results under multiplex PCR conditions. The primers used for the different STR markers and for sexing are shown in Table I. One of the primers was labelled (5' end) with a fluorescent dye (FAM, JOE or TAMRA; Biometra Gottlin, Germany) to enable the visualization and analysis of the PCR products. PCR amplification was performed in a total volume of 50 µl containing genomic DNA (15 µl of the extracted DNA), 200 µmol/l dNTPs, 4–20 pmoles of each primer, PCR buffer (Perkin Elmer Applied Biosystems Inc, USA) and 1.0 IU AmpliTaq Gold (Perkin Elmer Applied Biosystems Inc). Four separate multiplex PCR assays were designed using the STR markers (see Table II). After the initial denaturation at 94°C for 5 min, 31 cycles of PCR amplification followed (1 min denaturation at 94°C, 1 min annealing at 60°C, 1 min extension at 72°C, final extension for 5 min at 72°C.) and performed in a Gene Amp. PCR system 9700 cycler (Perkin Elmer Applied Biosystems Inc). The allelic fragments were resolved on a 4.25% denaturing polyacrylamide gel using a 377 DNA Sequencer (Perkin Elmer Applied Biosystems Inc). The Genescan 672 software was employed for the analysis and calculation of the amplification products. The study design was approved by the regional ethics committee.
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Results
A total of 662 amniotic fluid samples were analysed by QF–PCR (Table III). Of them, 586 (88.5%) were clear samples, whereas 39 (5.9%) were light and 37 (5.6%) dark red stained. No sample failed and had to be rerun. Males were diagnosed by amplification of the X and Y chromosomal PCR products of the amelogenin locus with a normal 1:1 ratio, females by the presence of one signal of the X chromosome PCR product. For the rapid diagnosis of trisomy 21 we used up to three STR markers, which resulted in an informative PCR pattern in 98.3% (651/662) of all analysed specimens. 97.3% (643/662) of all samples were heterozygous and thus informative for at least one STR marker specific for chromosome 18. Using up to three STR markers specific for chromosome 13, 94.6% (626/662) of all samples were found to be informative for at least one STR marker.
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The criteria and guidelines for the diagnosis of a normal or pathological QF–PCR result were as follows: for the diagnosis of a Turner syndrome a monoallelic pattern of all X-specific STR markers and the absence of the Y specific amelogenin locus was necessary (Figure 1
). Due to the heterozygosity indices (Table I
), the probability of homozygosity for each X chromosome marker is extremely low. A normal autosomal QF–PCR product was assumed after at least one STR marker for each chromosome displayed a clear diallelic pattern with a normal 1:1 ratio. The analysis of samples from patients with a trisomy revealed the presence of either three peaks (ratio 1:1:1) or two peaks with a ratio of 2:1 for the chromosome specific STR marker (Figure 2). In our laboratory we assumed a tolerance of ±20% for the calculation of the peak ratio. This calculation is necessary due to the PCR-specific worse preferential amplification of larger PCR fragments. Under these conditions, no preferential amplification leading to misdiagnosis was observed. One amniotic fluid which was dark red stained showed an abnormal (no 2:1 ratio) diallelic PCR pattern in two STR profiles and an unclear triallelic (three peaks which showed no 1:1:1 ratio) PCR pattern in five STR profiles. This sample was suspected of being contaminated and was classified as an artificial PCR product probably due to maternal contamination (Figure 3). The PCR products were not compared with maternal ones. Chromosomal analysis of this sample revealed a normal karyotype. Whenever a QF–PCR product was pathological, the result was validated within 24 h by QF–PCR with a second independent analysis of 1–2 ml uncultured amniotic fluid of the same sample. In all cases, the pathological results of the QF–PCRs were consistent with the cytogenetic analysis [45,X (n = 4); 47,XXY (n = 1); 69,XXX or 69,XXY (n = 3), 47,XX, or XY,+18 (n = 4); 47,XX, or XY,+21 (n = 5)]. One sample with a sex chromosome mosaicism (mos 45,X/46,XX; level III mosaicism) was misdiagnosed as an XX chromosome complement by QF–PCR. One case diagnosed as XXY status by QF–PCR displayed sex chromosome mosaicism (47,XXY/46,XX; level III mosaicism) by conventional chromosome analysis. No information was obtained in one fetal sample with a trisomy 18 due to an unclear 2:1 ratio for two of three applied STRs specific for chromosome 18. The third STR marker was uninformative. Two cases with an unbalanced Robertsonian translocation [46,XY,der(13;14) (q10;q10),+13; 46,XX,i(21) (q10)] could be identified by QF–PCR as trisomic for chromosomes 13 and 21 respectively. No false positive QF–PCR results were observed. Seven aberrant chromosome complements were not detectable by molecular analysis: After karyotyping, two cases showed an unbalanced karyotype [46,XX, in-situ hybridzation del(22)(q11.2); mosaic 47,XY,+20/46,XY] and five samples a balanced chromosome aberration (45,XX,der(13;14)(q10;q10) de novo; 45,XX,der(13;14) (q10;q10)pat; 46,XX,t(3;8)(p34.1;q25)mat; 46,XY,inv(9)(p24q32) pat; 46,XY, t(1;17) (p34.1;q25)mat.
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Discussion
The study presents the results of the first large clinical application of QF–PCRs for the rapid detection of sex chromosome and autosomal aneuploidies for chromosomes X and Y, 13, 18 and 21 on 662 uncultured clear and visibly contamined amniotic fluids as an adjunctive test to conventional cytogenetic analysis. Only one sample showed an artificial PCR pattern after applying all selected chromosome specific STR markers. In all other specimens, the high level of heterozygositiy of the X-specific STR markers SBMA, DXS8377 and DXS1283E in combination with the X/Y homologous gene amelogenin locus resulted in an informative PCR result. In normal female samples there was no single case of homozygosity for all applied X-linked markers. Turner syndrome (45,X, n = 4) was correctly identified by QF–PCR, showing only single SBMA, DXS8377 and/or DXS1283E peaks of fluorescence activity. In two cases, an XXY status could also be diagnosed by the assay. In one of these samples chromosome analysis revealed a sex chromosome mosaicism (47,XXY/46,XX), whereas the QF–PCR test was not able to detect the normal cell population. QF–PCR results leading to false negative results were found in only one case with mosaicism of the X chromosome (mosaic 45,X/46,XX). Although the QF–PCR products showed clear heterozygous STR profiles for DXS8377 and DXS1283 respectively, cytogenetic analysis yielded a sex chromosome mosaicism with the presence of a second population of cells with an X karyotype. False positive QF–PCR results were not observed. Within the current study, a total of 29 chromosomal abnormalities was diagnosed by cytogenetic analysis. Five of them showed a balanced chromosomal complement, mainly due to a familial Robertsonian or reciprocal translocation or inversion. Of the 24 unbalanced chromosome complements with a significant risk of phenotypic abnormalities, 20 (83%) were identified by QF–PCR.
FISH on uncultured amniotic cells is an alternative rapid method to QF–PCR. However, our results showed that the QF–PCR based diagnostic method has many advantages over FISH. As shown by several groups contamination by maternal blood was shown to pose a problem for aneuploidy detection by FISH. Two groups (Ward et al., 1993; Jalal et al., 1998) reported promising FISH data (only 2.3%, e.g. 0.3% of their samples failed), whereas Bryndorf et al. (1997) were not able to reproduce their results and Eiben et al. (1998, 1999) excluded all specimens appearing bloody or brownish. However, in 98.7% of all our samples (75/76) suspected of being contaminated, we did not find an abnormal STR pattern. Therefore, the potential risk of misdiagnosis in blood stained amniotic fluids seems to be very low by QF–PCR, thereby increasing the detection rate of chromosome aberrations, which is most important for the assay efficiency and consequently for the clinical utility. Moreover, in those of our cases where a contamination could be suspected, STR profiles of the fetal and the corresponding maternal blood sample could provide valid and accurate information about potential maternal contamination so that a correct interpretation is possible. Furthermore, 1–2 ml of uncultured amniotic fluid is sufficient for the QF–PCR analysis of aberrations of the sex chromosomes and all three autosomal chromosomes. FISH with a maximum probe set of five different chromosomes tests (using commercially available DNA probes) usually requires 2–5 ml of amniotic fluids (Eiben et al., 1998; Jalal et al., 1998). In our laboratory, the QF–PCR technique with one automatic DNA scanner and one technician allows the investigation of 18 amniotic fluid samples within 8 h. This is not possible with the FISH procedure: After the harvesting procedure and standard hybridization for a minimum of 6 h (Jalal et al., 1998), each FISH analysis requires a time intensive individual microscopic procedure (0.5 h per case).
In summary the study demonstrates the feasibility of accurate diagnosis of Turner syndrome, other common fetal sex chromosome and autosomal aneuploidies by QF–PCR within 1 day which is not possible by standard cytogenetic methods. Altogether the QF–PCR method is rapid and reliable, and we believe that it will be more efficient than alternative methods.
Acknowledgments
The authors thank C.Böttcher, K.Gey, Y.Grossmann, N.Reuter, and C.Schomburg for technical assistance. Finally, we thank the many patients and all gynaecologists who participated in these studies. W.Schmidt developed the molecular technique and carried out most of the practical work. J.Jenderny initiated the study and summarized the data. We received most of the pathological cases from B.J.Hackelöer and K.Hecher. L.Kochhan acted as a substitute for W.Schmidt. J.Jenderny and S.Kerber handled the cytogenetic analysis and K.R.Held supervised the project overall.
Notes
3 To whom correspondence should be addressed at: Institute of Immunology, Pathology, Molecular Biology and Human Genetics (IPM), Lademannbogen 61–63, 22339 Hamburg, Germany. E-mail: jenderny{at}mail.labor-keeser-arndt.de 
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Submitted on February 8, 2000; accepted on June 14, 2000.
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