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Volume 53 (3): 285-288, 2005
Copyright ©The Histochemical Society, Inc.
BRIEF REPORT |
Rapid Prenatal Diagnosis of Aneuploidy Using Quantitative Fluorescence-PCR (QF-PCR)
Cytogenetics Department, Guy's & St Thomas' Hospital Trust, London, UK
Correspondence to: Dr. Caroline Mackie Ogilvie, Cytogenetics Department, 5th Floor, Guy's Tower, St Thomas Street, London SE1 9RT, UK. E-mail: caroline.ogilvie{at}genetics.kcl.ac.uk
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Summary |
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 Top Summary Literature Cited |
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Molecular cytogenetic aneuploidy testing for pregnant women at increased risk of chromosome abnormality leads to rapid reassurance for those with normal results and earlier decisions on pregnancy management in the case of abnormality. We tested 9080 prenatal samples using a one-tube QF-PCR test for trisomies 13, 18, and 21; the abnormality rate was 5.9%. There were no misdiagnoses for non-mosaic trisomy. A sex chromosome multiplex was developed that detects structural sex chromosome abnormalities as well as aneuploidies. The sex chromosome test was targeted at pregnancies (272) with specific abnormalities suggestive of Turner syndrome; 13.2% showed 45,X, confirmed by follow-up analysis. (J Histochem Cytochem 53:285–288, 2005)
Key Words: prenatal diagnosis • QF-PCR • aneuploidy • Down syndrome • rapid testing • sex chromosome aneuploidy • Turner syndrome
WOMEN WITH PREGNANCIES at increased risk of chromosome abnormality (usually because of maternal age, altered serum metabolites, or ultrasound abnormalities of the fetus) undergo invasive sampling of either amniotic fluid (AF), chorionic villi (CVS) or, rarely, fetal blood. Material from these samples is cultured to obtain dividing cells and then harvested and prepared for full karyotype analysis of metaphase chromosomes. In the UK, the average reporting time for karyotype analysis of prenatal samples is about 14 days (NEQAS 2000
). The emergence of new molecular cytogenetic technology, first fluorescence in situ hybridization (FISH) in 1994 (Spathas et al. 1994) and then QF-PCR (Mansfield 1993), first established in the UK National Health Service (NHS) in 2000 (Mann et al. 2001), allowed 24–48 hr diagnosis of the most prevalent chromosome abnormalities (trisomy 13, trisomy 18, and trisomy 21) (Figures 1A and 1B), thus providing rapid reassurance for those women with normal results and early decisions on pregnancy management for abnormal fetuses. Testing for sex chromosome aneuploidy, aimed at detection of Turner syndrome (45,X), is incorporated into these rapid tests by some centers (Cirigliano et al. 1999; Levett et al. 2001), although others have chosen to target this test only to those pregnancies referred with abnormalities suggestive of 45,X (nuchal thickening >5 mm, nuchal risk of >1:5, cystic hygroma, hydrops, or specific cardiac abnormalities) (Donaghue et al. 2003). We present here a novel QF-PCR multiplex for detection of sex chromosome aneuploidy and our data on over 9000 pregnancies tested using QF-PCR.
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Preparation of samples, formulation of the trisomy multiplex, protocol for QF-PCR, and interpretation of results are as described previously (Mann et al. 2004
). Table 1 shows the primer sequences and target loci of the modified sex chromosome multiplex.
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All prenatal samples received in this laboratory undergo rapid trisomy testing. Table 2 shows the results from the trisomy multiplex during the period from June 2000 to March 2004. All samples had full karyotype analysis following QF-PCR testing, and there were no misdiagnoses for non-mosaic trisomy. There were 17 mosaic samples detected by QF-PCR and/or karyotype analysis. Of these, QF-PCR identified the abnormal cell line in 13 samples, and karyotype analysis identified the abnormal cell line in 14 samples. We estimate that QF-PCR can identify mosaicism at levels greater than 15%. The current trisomy multiplex is more informative than previous versions, leading to a greatly reduced incidence of uninformative results: in 2003–2004, there was only one sample with one uninformative chromosome. Maternal cell contamination (MCC) was evident as a second genotype but with no fourth allele and with allele peaks in characteristic ratios (Mann et al. 2001
). Almost all samples showing MCC were bloodstained amniotic fluid samples. Where the second genotype was low level and did not result in allele peak ratios outside the normal range for the majority genotype, results were interpreted and reported. If allele peak ratios were outside the normal range, samples were reported as "unsuitable."
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Since the introduction of a separate sex chromosome multiplex, 7794 prenatal samples (1753 CVS and 6041 AF samples) have been received and, of these, 272 (187 CVS and 85 AF samples) had referrals indicative of 45,X and were tested using either the sex chromosome multiplex previously published (Donaghue et al. 2003
) or the modified multiplex described here (Figures 1C and 1D). Of the 187 CVS tested, 29 (15.5%) had QF-PCR traces indicative of monosomy X; of the 85 AF samples tested, 7 (8.2%) were indicative of monosomy X. These results were confirmed by FISH analysis prior to reporting and were consistent with the follow-up karyotype analysis. All but two 45,X samples in this sample set were detected by the application of these selection criteria for testing. The two that did not fulfil the selection criteria were referred with nuchal measurements of 4.6 mm and 2.9 mm. No structural abnormalities or other sex chromosome aneuploidies were detected by the QF-PCR assay.
Our center applies sex chromosome testing only to pregnancies referred with characteristics suggestive of 45,X (Turner syndrome). This is because QF-PCR with primers for loci on the sex chromosomes will, in addition to 45,X, detect other sex chromosome aneuploidies such as 47,XXY (Klinefelter syndrome) and 47,XYY. These aneuploidies are associated with mild clinical phenotype and establishing their presence in pregnancies is of debatable value. In previously published multiplexes, the presence of the Y chromosome has been established by amplification of the amelogenin locus (Cirigliano et al. 1999; Donaghue et al. 2003). However, deletion of this locus in a proportion of the population has been reported (Steinlechner et al. 2002) with associated possibilities of misdiagnosis of a normal male as a Turner syndrome female. The multiplex reported here has, in addition to primers on the short and long arms of the X chromosomes, primers for the SRY locus on the short arm of the Y chromosome and two loci on the long arm and, thus, has the potential to diagnose structural abnormalities of the Y chromosome, such as i(Yp). In addition, there are other sex chromosome abnormalities, such as i(Xq), which are associated with a phenotype similar to Turner syndrome. The sex chromosome multiplex described here will detect i(Xq) and other structural abnormalities.
The targeted approach to sex chromosome testing (Donaghue et al. 2003) detected all but two cases of non-mosaic 45,X in the sample set reported here. All rapid results indicating 45,X were confirmed by FISH due to the small possibility (1:907) (Donaghue et al. 2003) that the result may represent a normal female with X chromosomes homozygous for the markers tested. The QF-PCR trace from such a case would be indistinguishable from a 45,X female. In the samples not targeted for QF-PCR sex chromosome testing, karyotype analysis found two cases of 47,XXX, four cases of 47,XXY, one case of 47,XYY, one balanced (X;8) translocation, one unbalanced (X;9) translocation, and one terminal deletion of the long arm of the X chromosome. It has been suggested that QF-PCR could be used as a stand-alone test for women referred for prenatal testing because of an increased risk of Down syndrome (Leung et al. 2003). In the event of this change of policy, a rapid test that did not detect chromosome abnormalities of unknown or negligible significance (such as 47,XYY) would alleviate counseling difficulties and parental anxiety.
QF-PCR for the detection of common chromosomal trisomies and sex chromosome aneuploidy has been introduced as a validated service into a number of European centers (Schmidt et al. 2000; Cirigliano et al. 2001; Levett et al. 2001; Mann et al. 2001). These centers use different multiplexes and different approaches to sex chromosome testing; however, QF-PCR appears to be a robust generic approach to rapid prenatal diagnosis and has advantages over other published approaches (Mann et al. 2004). The data set presented here confirms that QF-PCR is reliable, accurate, and robust, with no misdiagnoses for non-mosaic trisomy 13, 18, or 21, or triploidy.
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Acknowledgments |
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The authors thank the other members of the prenatal team for their contribution and Alysia Hallam for technical assistance.
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Footnotes |
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Presented in part at the 14th Workshop on Fetal Cells and Fetal DNA: Recent Progress in Molecular Genetic and Cytogenetic Investigations for Early Prenatal and Postnatal Diagnosis, Friedrich-Schiller-University, Jena, Germany, April 17–18, 2004.
Received for publication May 18, 2004; accepted June 22, 2004
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Literature Cited |
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TopSummaryLiterature Cited |
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Cirigliano V, Ejarque M, Canadas MP, Lloveras E, Plaja A, Perez MM, Fuster C, et al. (2001) Clinical application of multiplex quantitative fluorescent polymerase chain reaction (QF-PCR) for the rapid prenatal detection of common chromosome aneuploidies. Mol Hum Reprod 7:1001–1006
Cirigliano V, Sherlock J, Conway G, Quilter C, Rodeck C, Adinolfi M (1999) Rapid detection of chromosomes X and Y aneuploidies by quantitative fluorescent PCR. Prenat Diagn 19:1099–1103[CrossRef][Medline]
Donaghue C, Roberts A, Mann K, Ogilvie CM (2003) Development and targeted application of a rapid QF-PCR test for sex chromosome imbalance. Prenat Diagn 23:201–210[CrossRef][Medline]
Edwards A, Civitello A, Hammond HA, Caskey CT (1991) DNA typing and genetic mapping with trimeric and tetrameric tandem repeats. Am J Hum Genet 49:746–756[Medline]
Edwards A, Hammond HA, Jin L, Caskey CT, Chakraborty R (1992) Genetic variation at five trimeric and tetrameric tandem repeat loci in four human population groups. Genomics 12:241–253[CrossRef][Medline]
Leung WC, Lau ET, Lao TT, Tang MH (2003) Can amnio-polymerase chain reaction alone replace conventional cytogenetic study for women with positive biochemical screening for fetal Down syndrome? Obstet Gynecol 101:856–861
Levett LJ, Liddle S, Meredith R (2001) A large-scale evaluation of amnio-PCR for the rapid prenatal diagnosis of fetal trisomy. Ultrasound Obstet Gynecol 17:115–118[CrossRef][Medline]
Mann K, Donaghue C, Fox SP, Docherty Z, Ogilvie CM (2004) Strategies for the rapid prenatal diagnosis of chromosome aneuploidy. European Journal of Human Genetics 12:907–915[CrossRef][Medline]
Mann K, Fox SP, Abbs SJ, Yau SC, Scriven PN, Docherty Z, Ogilvie CM (2001) Development and implementation of a new rapid aneuploidy diagnostic service within the UK National Health Service and implications for the future of prenatal diagnosis. Lancet 358:1057–1061[CrossRef][Medline]
Mansfield ES (1993) Diagnosis of Down syndrome and other aneuploidies using quantitative polymerase chain reaction and small tandem repeat polymorphisms. Hum Mol Genet 2:43–50
NEQAS (2000) National External Quality Assessment Scheme in Clinical Cytogenetics. Annual Report
Schmidt W, Jenderny J, Hecher K, Hackeloer BJ, Kerber S, Kochhan L, Held KR (2000) Detection of aneuploidy in chromosomes X, Y, 13, 18 and 21 by QF-PCR in 662 selected pregnancies at risk. Mol Hum Reprod 6:855–860
Spathas DH, Divane A, Maniatis GM, Ferguson-Smith ME, Ferguson-Smith MA (1994) Prenatal detection of trisomy 21 in uncultured amniocytes by fluorescence in situ hybridization: a prospective study. Prenat Diagn 14:1049–1054[Medline]
Steinlechner M, Berger B, Niederstatter H, Parson W (2002) Rare failures in the amelogenin sex test. Int J Legal Med 116:117–120[CrossRef][Medline]
Sullivan KM, Mannucci A, Kimpton CP, Gill P (1993) A rapid and quantitative DNA sex test: fluorescence-based PCR analysis of X-Y homologous gene amelogenin. Biotechniques 15:636–638, 640[Medline]
Wehnert M, Reiner O, Caskey CT (1993) Four STR polymorphisms map to a 500 kb region between DXS15 and DXS134. Hum Mol Genet 2:1503
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