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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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 2448 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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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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Footnotes |
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Received for publication May 18, 2004; accepted June 22, 2004
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Literature Cited |
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