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Annals of the New York Academy of Sciences 12/2006; 731(1):186 - 192. · 3.15 Impact Factor
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L Rickman,
H Fiegler,
C Shaw-Smith,
R Nash,
V Cirigliano,
G Voglino,
B L Ng,
C Scott,
J Whittaker, M Adinolfi,
N P Carter,
M Bobrow
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ABSTRACT: Karyotype analysis has been the standard method for prenatal cytogenetic diagnosis since the 1970s. Although highly reliable, the major limitation remains the requirement for cell culture, resulting in a delay of as much as 14 days to obtaining test results. Fluorescent in situ hybridisation (FISH) and quantitative fluorescent PCR (QF-PCR) rapidly detect common chromosomal abnormalities but do not provide a genome wide screen for unexpected imbalances. Array comparative genomic hybridisation (CGH) has the potential to combine the speed of DNA analysis with a large capacity to scan for genomic abnormalities. We have developed a genomic microarray of approximately 600 large insert clones designed to detect aneuploidy, known microdeletion syndromes, and large unbalanced chromosomal rearrangements.
This array was tested alongside an array with an approximate resolution of 1 Mb in a blind study of 30 cultured prenatal and postnatal samples with microscopically confirmed unbalanced rearrangements.
At 1 Mb resolution, 22/30 rearrangements were identified, whereas 29/30 aberrations were detected using the custom designed array, owing to the inclusion of specifically chosen clones to give increased resolution at genomic loci clinically implicated in known microdeletion syndromes. Both arrays failed to identify a triploid karyotype. Thirty normal control samples produced no false positive results.
Analysis of 30 uncultured prenatal samples showed that array CGH is capable of detecting aneuploidy in DNA isolated from as little as 1 ml of uncultured amniotic fluid; 29/30 samples were correctly diagnosed, the exception being another case of triploidy. These studies demonstrate the potential for array CGH to replace conventional cytogenetics in the great majority of prenatal diagnosis cases.
Journal of Medical Genetics 05/2006; 43(4):353-61. · 6.36 Impact Factor
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V Cirigliano,
G Voglino,
M P Cañadas,
A Marongiu,
M Ejarque,
E Ordoñez,
A Plaja,
M Massobrio,
T Todros,
C Fuster,
M Campogrande,
J Egozcue, M Adinolfi
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ABSTRACT: The quantitative fluorescent PCR (QF-PCR) assay, introduced during the last few years, allows prenatal diagnoses of common chromosome aneuploidies in a few hours after sampling. We report the first assessment of QF-PCR performed on a large cohort of 18,000 consecutive clinical specimens analysed in two different Centres. All samples were analysed by QF-PCR using several selected STR markers together with amelogenin and, occasionally, SRY for fetal sexing. Results were compared with those obtained by conventional cytogenetic analysis. In 17,129 tests, normal fetuses were detected by QF-PCR. No false positives were observed. All 732 cases of trisomy 21, 18, 13, triploidies, double trisomies as well as all but one fetuses with X and Y aneuploidies were correctly diagnosed. Chromosome mosaicism could also be suspected in several samples. In some cases of in vitro culture failures, QF-PCR was the only evidence of fetal X, Y, 21, 18 and 13 chromosome complement. QF-PCR proved to be efficient and reliable in detecting major numerical chromosome disorders. The main advantages of the molecular assay are its very low cost, speed and automation enabling a single operator to perform up to 40 assays per day. QF-PCR relieves anxiety of most parents within 24 h from sampling and accelerates therapeutic interventions in the case of an abnormal result. In countries where large scale conventional cytogenetics is hampered by its high cost and lack of technical expertise, QF-PCR may be used as the only prenatal diagnostic test.
Molecular Human Reproduction 12/2004; 10(11):839-46. · 3.85 Impact Factor
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ABSTRACT: Rapid prenatal diagnoses of major chromosome aneuploidies have been achieved successfully using quantitative fluoresent PCR (QF-PCR) assays and small tandem repeat (STR) markers. Here we report the results of evaluating the use of previously untested X-linked STRs, (DXS6803) and (DXS6809), together with modified amelogenin (AMXY) sequences and the X22 marker that maps in the pseudoautosomal region PAR2 on the long arm of the X and Y chromosomes. These markers will allow prenatal diagnoses of sex chromosome aneuploidies such as 45,X (pure Turner Syndrome), 47,XXY and 47,XYY, while assessing the sex of the fetuses. Data are also presented concerning the difficulties associated with the evaluation of the frequencies of the various types of sub-populations of cells in amniotic fluid samples collected from fetuses with sex chromosome mosaicism. The results of evaluating the use of new markers for the rapid diagnosis of aneuploidies affecting chromosomes 21,18 and 13 are also presented. Three chromosome 21 specific STRs have been found to produce trisomic triallelic or diallelic patterns from all amniotic samples retrieved from fetuses with Down Syndrome. Since all samples tested were amplified and no false positive or negative results were observed, the present results confirm the diagnostic value of QF-PCR for the prenatal detection of major numerical chromosome disorders.
Annals of Human Genetics 10/2001; 65(Pt 5):421-7. · 2.57 Impact Factor
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The Lancet 10/2001; 358(9287):1030-1. · 38.28 Impact Factor
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ABSTRACT: Several investigations are in progress with the aim of performing prenatal diagnosis of inherited disorders by noninvasive or minimally invasive techniques. The most important approaches are based on the detection of fetal nucleated cells in maternal blood, the analysis of fetal DNA present in maternal plasma, and the identification and isolation of fetal trophoblastic cellular elements shed into the uterine cavity and the endocervical canal. In this review, we discuss the methods that have been employed for the collection of the transcervical samples at an early stage of gestation and the techniques used for the identification of fetal cells. We also report the results of using endocervical cells for the detection of fetal chromosomal disorders by fluorescent in-situ hybridization and for performing prenatal diagnosis of fetal Rh(D) phenotypes. Recent investigations have also shown that--after the isolation of trophoblastic cells from maternal contaminants by micromanipulation--transcervical samples can be employed for the prenatal diagnosis of single gene defects, such as those causing thalassemia and sickle cell anemia. Although the present results are promising, further investigations are required to demonstrate the feasibility of performing accurate diagnosis of fetal diseases by this minimally invasive approach in all transcervical samples retrieved at an early stage of gestation.
Journal of Human Genetics 02/2001; 46(3):99-104. · 2.57 Impact Factor
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ABSTRACT: To develop a new method of RhD/d genotype determination using a quantitative fluorescent PCR (QF-PCR) assay.
Polymerase chain reaction amplification (PCR) of fragments of exon 7 of both the RHD and RHCE genes was performed from 32 amniotic fluid and 26 chorionic villus samples known to be heterozygous for the RHD gene, 74 peripheral blood samples of RhD-positive blood donors (homozygous or heterozygous) estimated by serologic typing and 24 RhD-negative fetal samples. The number of copies of the RHD gene in RhD-positive samples was determined by comparing the fluorescent intensities of the amplification products specific for the RHD and the RHCE genes.
A ratio of fluorescent intensities of 1:1 clearly indicated D/D homozygous individuals whereas a ratio of 1:2 was demonstrated in samples from D/d heterozygous individuals. The mean fluorescent intensity ratio of the peak areas of homozygous samples was 1.12 (SD 0.128), the mean ratio of the peak areas of heterozygous samples was 0.51 (SD 0.060). Complete agreement was obtained between RhD/d typing by QF-PCR and RhD genotypes assessed by family studies and serological methods.
The fluorescent PCR-based DNA test allows easy, rapid and accurate determination of the zygosity for the RHD gene. This new technique provides useful information for the clinical management of pregnancies of sensitised RhD-negative mothers.
BJOG An International Journal of Obstetrics & Gynaecology 01/2001; 107(12):1498-502. · 3.41 Impact Factor
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Journal of Medical Genetics 06/2000; 37(5):E1. · 6.36 Impact Factor
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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.
Molecular Human Reproduction 01/2000; 5(12):1176-9. · 3.85 Impact Factor
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ABSTRACT: Quantitative fluorescent polymerase chain reaction (QF-PCR) assays and small tandem repeat (STR) markers have been successfully employed for the rapid detection of major numerical aneuploidies affecting human autosomes. So far, the analysis of chromosomes X and Y disorders has been hampered by the rarity of highly polymorphic markers which could distinguish normal female homozygous PCR patterns from those seen in patients with Turner's syndrome. A new marker (X22) of the X/Y chromosomes has been identified which maps in the Xq/Yq pseudoautosomal region PAR2; used together with the HPRT it allows the rapid diagnosis of numerical aneuploidies of the sex chromosomes. Blood samples from normal male and female subjects and from patients with X and Y chromosome disorders (45,X and 47, XXY) have been tested by QF-PCR with the X22 polymorphic pentanucleotide (12 alleles) together with the HPRT and P39 markers. The samples were also tested by multiplex QF-PCR with STRs specific for chromosomes 21,18,13 and amelogenin (AMXY). Tested by QF-PCR, all samples from normal females were heterozygous for either the X22 or the HPRT marker with fluorescent peak ratios near 1:1, thus allowing a correct, rapid diagnosis of their chromosome complement. Turner's patients (45,X) showed only one X22 and one HPRT fluorescent peak, thus documenting the presence of a single X chromosome. Turner's patients with mosaicism showed a major fluorescent peak for the X22 and HPRT markers and a minor peak revealing the presence of a second minor population of cells. Two 47, XXY cases could also be diagnosed. Multiplex analyses can be performed using simultaneously STR markers for chromosomes 21,18,13 X and Y. The diagnostic value of a third X-linked marker (P39) was also investigated. These results suggest that rapid diagnosis of major numerical anomalies of the X and Y chromosomes can be performed using QF-PCR with a new highly polymorphic X-linked marker, X22, which maps in the Xq/Yq pseudoautosomal region PAR 2. Multiplex QF-PCR tests-using the X22 STR in association with HPRT and, in rare cases, a third P39 marker-allow the rapid diagnosis of major aneuploidies affecting chromosomes 21, 18, 13, X and Y. The X22 marker can also be employed for the detection of fetal cells present in maternal peripheral blood or the endocervical canal.
Prenatal Diagnosis 01/2000; 19(12):1099-103. · 2.11 Impact Factor
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ABSTRACT: Prenatal diagnoses of haemoglobin (Hb) mutations were performed using transcervical cells, retrieved by aspiration from the endocervical canal of ten selected pregnant women at about 10 weeks of gestation, prior to chorionic villus sampling (CVS). Both parents were carriers of haemoglobinopathies (thalassaemia or HbS). Clumps of fetal cells were isolated by micromanipulation under an inverted microscope and aliquots of the extracted DNA tested separately for the presence of paternally derived chromosome markers and Hb mutations by quantitative fluorescent polymerase chain reaction (PCR). The correct prenatal diagnosis of Hb diseases, using selected single clumps of trophoblastic cellular elements free of maternal contaminating cells, was achieved in six out of ten cases.
Clinical Genetics 12/1999; 56(5):357-61. · 3.13 Impact Factor
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ABSTRACT: We report the results of the first major study of applying quantitative fluorescence polymerase chain reaction (QF-PCR) assays for the detection of major chromosome numerical disorders. The QF-PCR tests were performed on a total of 247 chorionic villus samples, which were analysed blind, without any knowledge of the results obtained using conventional cytogenetic analysis. The aims of this investigation were to evaluate the detection power and accuracy of this approach by testing a large number of fetal samples and to assess the diagnostic value of each of the chromosome specific small tandem repeat (STR) markers used. In addition, we introduced three more markers specific for chromosomes 13, 18, and X to allow an accurate analysis of samples homozygous for a particular STR. Fluorescent labelled primers were used to amplify 12 STRs specific for chromosomes 21, 18, 13, X, and the amylogenin-like DNA sequence AMXY, expressed on the X and Y chromosomes. In this blind study of 247 fetal samples, 222 were correctly diagnosed by QF-PCR as normal for each of the five chromosomes investigated; 20 were diagnosed by QF-PCR as trisomic for chromosomes 21, 18, or 13, in agreement with the cytogenetic tests. Only one false negative result was observed, probably owing to the mishandling of the sample, which had been transferred through three laboratories before being analysed by QF-PCR. The 247 samples also included four cases of mosaicism or translocation; one case of mosaic trisomy 21 was detected by QF-PCR and the other cases were not identified by QF-PCR. The results of this investigation provide clear evidence that the QF-PCR assays are powerful adjuncts to conventional cytogenetic techniques and can be applied for the rapid and accurate prenatal diagnosis of the most frequent aneuploidies.
Journal of Medical Genetics 05/1999; 36(4):300-3. · 6.36 Impact Factor
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ABSTRACT: We have refined polymerase chain reaction (PCR) assays for the detection of sickle cell anaemia, the delta F 508 deletion causing cystic fibrosis, and the IVS1-110 mutation leading to beta thalassaemia, allowing them to be successfully performed upon single cells using fluorescent primers. We have also assessed the possibility of detecting aneuploidies of chromosomes 13, 18 and 21 using a quantitative fluorescent polymerase chain reaction (QF-PCR) with primers flanking polymorphic short tandem repeat (STR) markers. Trisomies were readily diagnosed by the detection of tri-allelic patterns. However some heterozygote normal and trisomic diallelic patterns did not produce the expected ratios of amplified PCR products due to preferential DNA sequence amplification. Total allelic drop out (ADO) did not occur with any of the cells tested. Multiplex QF-PCR assays can be performed on a single cell in under 6 h and simultaneously provide diagnosis of single gene defects, sex determination and an indication of selected chromosome aneuploidy.
Annals of Human Genetics 02/1998; 62(Pt 1):9-23. · 2.57 Impact Factor
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ABSTRACT: The incidence of chromosomal abnormalities in liveborn infants has been established to be about 1 in 170 newborns (1). The most frequent chromosomal anomalies are aneuploidies involving chromosomes 21,18,13, and both sex chromosomes. Prenatal diagnosis of chromosomal disorders is performed by conventional cytogenetic analysis of fetal cells collected by amniocentesis, chorionic villus sampling, or fetal blood sampling. Cytogenetic techniques allow detection of chromosome aneuploidies with great accuracy. The major disadvantage of these procedures is that fetal cells must be cultured for up to two weeks before analysis. This interval of time places a significant emotional and/or clinical burden on the parents and the physician. Moreover, if the fetus is abnormal and there is a potential need for therapeutic measures, a rapid answer is of great importance. Attempts to perform rapid prenatal diagnostic tests by fluorescent in situ hybridization (FISH) on interphase nuclei of amniocytes are still hampered by technical difficulties 2), Consequently, this approach has not yet entered into the realm of routine diagnostic procedures.
Methods in molecular medicine 01/1998; 16:287-99.
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ABSTRACT: Several studies have been performed to assess the diagnostic value of using small tandem repeat (STR) markers and quantitative fluorescent polymerase chain reaction (QF-PCR) assays for the rapid detection of aneuploidies involving chromosomes 21, 18, 13 (Mansfield, 1993; Pertl et al., 1994, 1996; Adinolfi et al., 1995a). The results of these investigations have documented the diagnostic advantages of this approach to perform prenatal tests using amniotic and chorionic samples, or fetal nucleated cells retrieved from peripheral maternal blood or endocervical samples. The use of two or more STR markers for each autosome facilitates the diagnosis of aneuploidies, while avoiding the need to employ internal non-polymorphic markers. Multiplex quantitative fluorescent analyses can be performed in about six hours from the collection of the samples and, although targeted to specific abnormalities, they can exclude the presence of the most frequent chromosomal disorders. QF-PCR can be exploited to analyse DNA present in single or clumps of cells and thus to perform prenatal diagnoses on maternal peripheral blood or transcervical cell samples and on preimplantation embryos.
Prenatal Diagnosis 12/1997; 17(13):1299-311. · 2.11 Impact Factor
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ABSTRACT: We have developed a quantitative fluorescence multiplex polymerase chain reaction assay for the rapid detection of sex and aneuploidies involving chromosomes 21, 18, and 13.
Samples of deoxyribonucleic acid (n = 85) extracted from amniotic fluid, fetal tissues, and blood were investigated by multiplex polymerase chain reaction amplification of polymorphic small tandem repeat markers specific for chromosomes 21, 18, 13, and X.
Quantitative analysis of the polymerase chain reaction products allowed us to distinguish between normal samples and samples with autosomal trisomies while sexing was performed simultaneously. From 85 samples only three produced unsatisfactory results with one of the two chromosome 13-specific markers. In these three cases the amplification of the other chromosome 13 marker always resulted in a correct normal pattern.
Quantitative fluorescence multiplex polymerase chain reaction is a reliable and rapid method that allows prenatal diagnosis of the major numeric chromosomal abnormalities to be performed within 24 hours.
American Journal of Obstetrics and Gynecology 11/1997; 177(4):899-906. · 3.47 Impact Factor
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ABSTRACT: Prenatal diagnoses were performed on six selected pairs of parents known to be carriers of Hb mutations by testing transcervical cells (TCCs) retrieved, prior to chorionic villus sampling (CVS), by aspiration of the cervical mucus from the pregnant mothers at 10-12 weeks of gestation. A concordance between the results of testing chorionic villus cells and isolated clumps of trophoblastic cellular elements was observed in four of the six cases.
Prenatal Diagnosis 07/1997; 17(6):539-43. · 2.11 Impact Factor
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ABSTRACT: In the course of an investigation aimed at detecting the presence of trophoblastic cells in the endocervical canal of pregnant women between 7 and 17 weeks of gestation, several cases of aneuploidies were observed using a fluorescent in situ hybridisation (FISH) assay. The cases include fetal chromosome 21 and 18 trisomies, triploidy and sex chromosome aneuploidies. The results were confirmed by testing placental tissues obtained after termination of pregnancy (TOP). In two of these cases, clumps of cells with the morphology of trophoblasts were isolated from the transcervical cell (TCC) samples using micromanipulation. FISH and fluorescent polymerase chain reactions (PCR), performed on these clumps, showed them to be exclusively of fetal origin. These results show that prenatal diagnoses of major aneuploidies can be performed by FISH using whole TCC samples, or on isolated clumps of cells by FISH and PCR assays.
Journal of Medical Genetics 05/1997; 34(4):302-5. · 6.36 Impact Factor
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ABSTRACT: Aneuploidies involving chromosomes 21, 18, 13, X and Y account for over 95% of all chromosomal abnormalities in live-born infants. Prenatal diagnosis of these disorders is usually accomplished by cytogenetic analysis of amniotic or chorionic cells but this is a lengthy procedure requiring great technical expertise. In this paper, we assess the diagnostic value of using a quantitative fluorescent polymerase chain reaction (PCR) suitable for the simultaneous and rapid diagnosis of trisomies 21 and 18 together with the detection of DNA sequences derived from the X and Y chromosomes. Samples of DNA, extracted from amniotic fluid, fetal blood or tissues, and peripheral blood from normal adults were investigated by quantitative fluorescent PCR amplification of polymorphic small tandem repeats (STRs) specific for two loci on each of chromosomes 21 and 18. Quantitative analysis of the amplification products allowed the diagnosis of trisomies 21 and 18, while sexing was performed simultaneously using PCR amplification of DNA sequences derived from the chromosomes X and Y. These results indicate the advantages of using two sets of STR markers for the detection of chromosome 21 trisomies and confirmed the usefulness of quantitative fluorescent multiplex PCR for the rapid prenatal diagnosis of selected chromosomal abnormalities.
Human Genetics 08/1996; 98(1):55-9. · 5.07 Impact Factor
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ABSTRACT: Trophoblastic cells can be retrieved from the endocervix and the lower uterine segment in early pregnancy by aspiration or lavage (Rodeck et al., 1995). The feasibility of using this technique for prenatal diagnosis depends on how frequently fetal cells can be retrieved and whether such cells can be purified from the predominant maternal cell population. In this study, specimens retrieved from the lower uterine segment prior to elective first-trimester termination of pregnancy were examined histologically and characterized using a panel of monoclonal antibodies in an avidin-biotin-peroxidase technique. Lavage samples generally contained fewer cervical epithelial cells than aspirates. Syncytial fragments or cytotrophoblast were identified in 9 of 12 lavage samples but in only 4 of 10 aspirates. Trophoblast cells were reactive with various anti-trophoblast monoclonal antibodies but the trophoblast cells present displayed considerable antigenic heterogeneity. For positive selection of trophoblast cells from these samples, it is likely that the best yield will be achieved by using a panel of carefully characterized monoclonal antibodies directed against various villous and extravillous trophoblast populations.
Prenatal Diagnosis 01/1996; 15(12):1143-53. · 2.11 Impact Factor
