A chromosome 21-specific cosmid cocktail for the detection of chromosome 21 aberrations in interphase nuclei

Fluorescent in situ hybridization (FISH) with a 21q11-specific probe (CB21c1) consisting of three non-overlapping cosmids has been applied to interphase amniocytes of pregnancies at increased risk for fetal aneuploidy (N = 78) and to interphase lymphocytes, cultured and uncultured, of patients referred for Down syndrome (N = 19 and 28, respectively). In the uncultured amniocytes, six chromosome aberrations were detected: three cases of trisomy 21, a triploidy, a de novo 46,XX,t(21q21q), and a mosaic 46,XY/47,XY,+dic(21)(q11)/48,XY,+dic(21)(q11), +del(21)(q11). In 15 cultured and 20 uncultured blood samples, FISH correctly diagnosed trisomy 21 (full or mosaic) at the interphase level, which was confirmed in all cases by subsequent karyotyping. Because of specific and strong signals in interphase nuclei, CB21c1 appears to be a useful tool for the rapid detection of chromosome 21 abnormalities.     

Prenatal detection of trisomy 21 in uncultured amniocytes by fluorescence in situ hybridization: A prospective study

A prospective study was undertaken to evaluate the use of fluorescence in situ hybridization (FISH) for the detection of trisomy 21 in interphase nuclei of uncultured amniotic fluid cells. Five hundred cases were analysed in situ and classified as normal or abnormal; the results were subsequently checked against the cytogenetic findings. Four hundred and ninety-three were correctly identified as normal with an 86·6 per cent average frequency of scored nuclei exhibiting two signals; six cases were correctly identified as trisomic for chromosome 21 with 81·7 per cent of scored nuclei exhibiting three signals; and one abnormal case involving an unbalanced chromosome 21·21 translocation was falsely scored as normal due to poor hybridization/detection efficiency. The method has been substantially improved and simplified so that it is suitable for the rapid detection of trisomy 21. As aneuploidy detection in interphase does not identify structural chromosome aberrations, it is not a substitute for fetal chromosome analysis.     

Rapid detection of numerical aberrations of chromosomes 13, 18 and 21 in chorionic mesenchymal cells

We have devised and evaluated a rapid screening method for the detection of numerical aberrations of chromosomes13, 18 and 21 in chorionic villus cells. We used non-radioactive in situ hybridization (ISH) with three chromosome-specific probes on overnight-attached mesenchymal cells from chorionic villi. A blind study was performed of 47 karyotypically normal samples, one triploid sample, two samples trisomic for chromosome 21, and two samples from a fetus with putative mosaicism (46/47, +21). All samples were hybridized with the chromosome 18- and 21-specific probes. Thirty samples were additionally hybridized with the chromosome 13-specific probe. The test could be completed within 3-4 days of sampling. In samples disomic with respect to the probed chromosomes, an average of 2 per cent (range 0-9 per cent) had three hybridization signals. By contrast, in the samples trisomic for the probed chromosome(s), 57 per cent (chromosome 13), 51 per cent (chromosome 18), and an average of 74 per cent (55-86 per cent) (chromosome 21) of the nuclei exhibited three signals. In the putative mosaic samples, the number of nuclei with three chromosome 21-specific signals ranged from 41 to 69 per cent. We conclude that this technique rapidly and clearly distinguishes between normal and trisomic/triploid samples, and consequently may be of use in future prenatal diagnosis.     

Prenatal diagnosis of the derivative chromosome 22 associated with cat eye syndrome by fluorescence in situ hybridization

Cytogenetic studies of cultured amniocytes demonstrated a karyotype of 46, XX/47, XX,+mar. A bisatellited, dicentric, distamycin-DAPI negative, NOR-positive marker was present in 76 per cent of the metaphases examined. Similar markers have been associated with cat eye syndrome (CES). We report on the utilization of fluorescence in situ hybridization (FISH) with a 14/22 a-satellite probe and a chromosome 22-specific cosmid for locus D22S9 to determine the origin of the prenatally detected supernumerary marker chromosome. FISH studies demonstrated that the marker is a derivative of chromosome 22 and enabled us to provide the family with additional prognostic information.     

Rapid prenatal diagnosis of trisomy 18 and triploidy in interphase nuclei of uncultured amniocytes by non-radioactive in situ hybridization

Two biotinylated chromosome-specific DNA probes were used to quantify the number of chromosomes 18 and 1 in uncultured amniocytes. Thirty-three samples of uncultured amniocytes were hybridized with a chromosome 18-specific DNA probe. Uncultured cells from two of the 33 samples were also hybridized with a chromosome 1-specific probe. Thirty of the samples were disomic with respect to chromosome 18; two samples were trisomic with respect to chromosome 18, and one sample was trisomic with respect to chromosomes 1 and 18. The two cases of trisomy 18 and the single case of triploidy were identified on uncultured celis within 48-72 h after amniocentesis. They were found among five samples from pregnant women who had amniocentesis because of an ultrasonographically identified fetal malformation. A trisomic karyotype could be diagnosed with certainty in uncultured amniocytes because the majority of the responding nuclei exhibited three hybridization signals. In normal cells, the majority of nuclei exhibited two signals. In no cases was there discordance between the genotype as predicted by in situ hybridization and that determined by cytogenetic analysis.     

Evaluation of X,Y, 18, and 13/21 alpha satellite DNA probes for interphase cytogenetic analysis of uncultured amniocytes by fluorescence in situ hybridization

The major aneuploidies diagnosed prenatally involve the autosomes 13, 18, and 21, and sex chromosomes. Fluorescence in situ hybridization (FISH) allows rapid analysis of chromosome copy number in interphase cells. This prospective study evaluated the use of four commercially available centromeric DNA probes (DXZ1, DYZ1, D18Z1, and D13Z1/D21Z1) for direct analysis of uncultured amniocytes. One hundred and sixteen amniotic fluid samples were analysed by FISH and standard cytogenetics. This evaluation demonstrated that FISH with, X, Y, and 18 alpha satellite DNA probes could accurately and rapidly detect aneuploidies involving these chromosomes and could be used in any prenatal clinical laboratory. In contrast, the 13/21 alpha satellite DNA probe hybridizing both chromosomes 13 and 21 was unreliable for prenatal diagnosis in uncultured amniocytes.     

Prenatal diagnosis by in situ hybridization on uncultured amniocytes: Reduced sensitivity and potential risk of misdiagnosis in blood-stained samples

Maternal cell contamination was assessed in 18 macroscopically blood-stained amniotic fluid samples from male fetuses. The samples were analysed by double-target fluorescent in situ hybridization (ISH) with Y and X chromosome-specific probes. The only sample with an aberrant karyotype (47, XY, +18) was also analysed by hybridization with a chromosome 18-specific probe. An interpretation of extensive maternal cell contamination was made in two samples, one of which was the sample with trisomy 18. ISH with the chromosome 18-specific probe on this latter sample showed that the sensitivity of the ISH method for chromosome enumeration of uncultured amniotic fluid samples may be reduced in bloodstained samples. It was calculated that by using ISH for chromosome enumeration of the two extensively contaminated samples, a case of trisomy 21 might have been overlooked in both samples, while a case of trisomy 18 might only have been overlooked in one of the samples. It is concluded that ISH should not be used for chromosome enumeration of uncultured amniotic fluid samples that are macroscopically blood-stained without further technical developments.     

Isolation of fetal cells from transcervical samples by micromanipulation: Molecular confirmation of their fetal origin and diagnosis of fetal aneuploidy

Transcervical cell (TCC) samples have been shown to contain fetal cells amenable to molecular analysis. However, the presence of ‘contaminating’ maternal cells limits their use for prenatal diagnoses. In this report we show that clumps of fetal cells can be isolated from transcervical samples by micromanipulation and tested by fluorescence in situ hybridization (FISH) and polymerase chain reaction (PCR). Out of 129 clumps, isolated from mucus aspirates and transcervical lavages from 29 patients, 29 clumps from 11 patients were found to be exclusively of fetal origin as judged by the detection of chromosome 21-specific polymorphic DNA markers and Y-derived DNA sequences by PCR and FISH. One case of a male triploid fetus, diagnosed by the analysis of TCC samples obtained by mucus aspiration and lavage, was confirmed by testing clumps of cells isolated by micromanipulation.     

Double locus analysis of chromosome 21 for preimplantation genetic diagnosis of aneuploidy

Preimplantation genetic diagnosis (PGD) of numerical chromosome abnormalities significantly reduces spontaneous abortions and may increase pregnancy rates in women of advanced maternal age undergoing in vitro fertilization. However, the technique has an error rate of around 10% and trisomy 21 conceptions have occurred after PGD. To further reduce the risk of transferring trisomy 21 embryos to the patient, we designed a protocol that analyzes chromosome 21 twice by targeting two different loci. This protocol was applied to 388 embryos from 60 cycles of PGD of aneuploidy. The scoring criterion used was based on giving equal importance to both probe results. Of the 242 embryos diagnosed as abnormal, 125 were re-biopsied to assess the rate of false positives and false negatives of the protocol and their clinical relevance. The results of the present study showed no reduction in the overall fluorescent in situ hybridization (FISH) error rate for single cells. However, by using a different scoring criterion, the incidence of false negative can be reduced to 1.6% without missing any trisomy 21. In addition, the present study suggests that if two or more loci from the same chromosome could be simultaneously analyzed in single cells, errors caused by false monosomies could be reduced. Copyright © 2001 John Wiley & Sons, Ltd.     

Rapid prenatal diagnosis of aneuploidy from uncultured amniotic fluid cells using five-colour fluorescence in situ hybridization

In this paper we describe the use of five-colour fluorescence in situ hybridization for prenatal diagnosis of aneuploidy using uncultured amniotic fluid cells. The analysis is based on ratio mixing of dual-labelled probes and digital imaging for the detection and visualization of five different probes specific for the five target chromosomes, 13, 18, 21, X, and Y. A retrospective blind analysis of 30 coded uncultured amniotic fluid samples correctly detected fetal sex and five trisomy 21 cases. Multicolour fluorescence in situ hybridization used in this way allows rapid and simultaneous detection of the most frequent aneuploidies.     

Role of amniotic fluid interphase fluorescence in situ hybridization (FISH) analysis in patient management

We retrospectively reviewed 309 amniotic fluid interphase fluorescence in situ hybridization (FISH) analyses performed from October 1995 to June 1999 to assess the role of interphase FISH in the management of patients at increased risk for fetal aneuploidies. Gestational age and indications for amniocentesis, clinical interventions after FISH results, as well as interventions after final culture reports were analyzed. There were 244 (79%) normal, 50 (16%) abnormal and 15 (5%) inconclusive FISH results. There were no false-positive or false-negative results, but there were nine (3%) clinically significant chromosomal abnormalities not detectable by FISH. Of the 50 women with abnormal FISH results, 26 (52%) elected to terminate the pregnancy prior to the availability of the standard chromosome analysis. In two of the fetuses with trisomy 21 no abnormalities were reported by ultrasound examination. Our experience indicates that interphase FISH results played an important role in decision making, especially for pregnancies close to 24 weeks' gestation. Standard karyotype analysis is still required for detection of chromosome abnormalities not detectable by interphase FISH techniques and for clarification of unusual or inconclusive FISH results. Copyright © 2001 John Wiley & Sons, Ltd.     

Parental mosaic trisomy 21 detected following maternal cell contamination of an amniotic fluid specimen from a normal male pregnancy

We report a case of maternal mosaic trisomy 21 ascertained at prenatal diagnosis as a result of maternal cell contamination of an amniotic fluid sample. A 34 year old female was referred for karyotyping because of a previous trisomy 21 pregnancy. Chromosome analysis of primary in situ cultures showed a karyotype of 47,XX, + 21[6]/46,XY[32]/46,XX[2]. Molecular testing demonstrated maternal cell contamination of the amniotic fluid sample and G-banded karyotyping of maternal blood showed that 3/200 cells had trisomy 21, consistent with the mother being a Down syndrome mosaic. A normal male baby with a 46,XY chromosome complement was delivered at 30 weeks. This case emphasises the need for close collaboration between cytogenetic and molecular genetics laboratories in resolving unusual cases of mosaicism. Copyright © 2007 John Wiley & Sons, Ltd.     

Familial X centromere variant resulting in false-positive prenatal diagnosis of monosomy X by interphase FISH

Interphase fluorescent in situ hybridization (FISH) analysis performed on uncultured amniotic fluid cells from a female fetus revealed a single signal using an X chromosome alpha-satellite probe, and the absence of any signal using a Y chromosome alpha-satellite probe. This result was initially interpreted as monosomy for the X chromosome in the fetus. Subsequent chromosome analysis from the cultured amniotic fluid cells showed two apparently normal X chromosomes. FISH using the X alpha-satellite probe on metaphase spreads revealed hybridization to both X chromosomes, although one signal was markedly reduced compared to the other. The same hybridization pattern was observed in the mother of the fetus. This is the first report of a rare familial X centromere variant resulting in a false-positive diagnosis of monosomy X by interphase FISH analysis for prenatal diagnosis. Copyright © 2001 John Wiley & Sons, Ltd.     

False-positive diagnosis of trisomy 21 using fluorescence in situ hybridisation (FISH) on uncultured amniotic fluid cells

An amniocentesis was performed on a 22-week pregnancy following the detection of foetal abnormalities on ultrasound. A rapid aneuploid screen using fluorescence in situ hybridisation on uncultured amniotic fluid cells revealed 3 signals for chromosome 21, consistent with trisomy 21. In situ metaphase cultures revealed a 46,XY normal male karyotype. These discordant results may be explained by a sub-standard batch of the commercially available probe or alternatively, a very specific variation within the sample interacting with the probe. Alternative strategies are proposed in order to safeguard against inappropriate clinical action as a consequence of discordant results. Copyright © 2003 John Wiley & Sons, Ltd.     

Prenatal diagnosis of partial monosomy 18p(18p11.2→pter) and trisomy 21q(21q22.3→qter) with alobar holoprosencephaly and premaxillary agenesis

A prenatal diagnosis of partial monosomy 18p(18p11.2→pter) and trisomy 21q(21q22.3→qter) in a fetus with alobar holoprosencephaly (HPE) and premaxillary agenesis (PMA) but without the classical Down syndrome phenotype is reported. A 27-year-old primigravida woman was referred for genetic counselling at 21 weeks' gestation due to sonographic findings of craniofacial abnormalities. Level II ultrasonograms manifested alobar HPE and median orofacial cleft. Cytogenetic analysis and fluorescence in situ hybridization (FISH) on cells obtained from amniocentesis revealed partial monosomy 18p and a cryptic duplication of 21q,46,XY,der(18)t(18;21)(p11.2;q22.3), resulting from a maternal t(18;21) reciprocal translocation. The breakpoints were ascertained by molecular genetic analysis. The pregnancy was terminated. Autopsy showed alobar HPE with PMA, pituitary dysplasia, clinodactyly and classical 18p deletion phenotype but without the presence of major typical phenotypic features of Down syndrome. The phenotype of this antenatally diagnosed case is compared with those observed in six previously reported cases with monosomy 18p due to 18;21 translocation. The present study is the first report of concomitant deletion of HPE critical region of chromosome 18p11.3 and cryptic duplication of a small segment of distal chromosome 21q22.3 outside Down syndrome critical region. The present study shows that cytogenetic analyses are important in detecting chromosomal aberrations in pregnancies with prenatally detected craniofacial abnormalities, and adjunctive molecular investigations are useful in elucidating the genetic pathogenesis of dysmorphism. Copyright © 2001 John Wiley & Sons, Ltd.     

Rapid karyotyping in prenatal diagnosis: A comparative study of the ‘pipette method’ and the ‘in situ’ technique for chromosome harvesting

Rapid karyotyping in the second and third trimesters has important implications for the management of pregnancies at risk. From September 1985 to March 1992, 735 amniotic fluid samples sent to our laboratory for rapid karyotyping from 64 different diagnostic centres of the Federal Republic of Germany were included in a comparative study on harvesting for chromosome analysis using the ‘pipette method’ or the ‘in situ’ technique. The average time between preparation of the amniotic fluid and verbal notification of the analysed karyotype was 5·41 days. The ‘pipette method’ needed on average 4·65 days, and the ‘in situ’ technique 5·97 days. In comparison with other more invasive techniques available for rapid karyotyping such as cordocentesis and placental biopsy, amniocentesis and subsequent chromosome harvesting using the ‘pipette method’ and/or the ‘in situ’ technique proved very useful and efficient. The overall incidence of chromosome aberrations was 15·3 per cent. The high rate of structural chromosome aberrations and uncommon aneuploidies found in our investigation (12 per cent) indicates that for rapid karyotyping in the second and third trimesters, conventional cytogenetic techniques cannot be replaced by faster techniques based on fluorescent in situ hybridization on interphase cells in the near future.     

Rapid prenatal diagnosis of 14 cases of triploidy using fish with multiple probes

Fluorescence in situ hybridization (FISH) of chromosome-specific probes to interphase nuclei can rapidly identify aneuploidies in uncultured amniotic fluid cells. Using DNA probe sets specific for chromosomes 13, 18, 21, X, and Y, we have identified 14 fetuses where the hybridization pattern was consistent with a triploid chromosome constitution. In each case, the identification of fetal abnormalities by ultrasound examination initiated a request for rapid determination of ploidy status via prenatal FISH analysis of uncultured amniocytes. FISH produced a three-signal pattern for the three autosomes in combination with signals indicating an XXX or XXY sex chromosome complement. This hybridization pattern was interpreted to be consistent with triploidy. Results were reported to the physician within 2 days of amniocentesis and subsequently confirmed by cytogenetics. These cases demonstrate the utility of FISH for rapid prenatal identification of triploidy, particularly when fetal abnormalities are seen with ultrasonographic examination.     

Amniotic fluid alpha-fetoprotein levels and prenatal diagnosis of autosomal trisomies

Pregnancies with fetal trisomy 21 have been associated with low amniotic fluid alpha-fetoprotein levels (AFAFP). This observation led to the suggestion that low AFAFP levels be used as a criterion for completion of a chromosomal analysis in patients who are not otherwise at increased risk for a fetal chromosome abnormality and in whom karyotyping might not have been completed for economic reasons. In order to assess the usefulness of such criteria, we reviewed the AFAFP levels of 90 cases of fetal trisomy 21, 23 cases of trisomy 18, and 10 cases of trisomy 13. These were compared with 2400 control samples with normal chromosome constitution. AFAFP levels were generally lower in pregnancies with trisomy 21, showing a median value of 0·72 MoM. However, 40 per cent of the trisomy 21 samples had AFAFP values greater than 0·8 MoM and 20 per cent were over 1·0 MoM. These data imply that over 50 per cent of Down syndrome cases might have been missed using a cut-off level of 0·70 MoM for completion of chromosome analysis. Using a higher cut-off level will leave only a small percentage of samples unkaryotyped. The distribution of AFP levels in trisomy 13 and 18 is no different from controls; we therefore believe that fetal karyotyping should be completed in every amniotic fluid sample obtained.     

Fluorescent in situ hybridization (FISH): A new application in the delineation of true vs. pseudomosaicism in prenatal diagnosis

Metaphase chromosomes and interphase nuclei from nine amniotic fluid cultures were studied with fluorescence in situ hybridization (FISH). The samples were initially analyzed with routine G-banding and were diagnosed as having true mosaicism (five patients) or pseudomosaicism (four patients). In our study, FISH analysis could provide additional information to distinguish pseudo– from true mosaicism by allowing interphase studies and analysis of an increased number of metaphase spreads. These results suggest a multilinear origin of ‘in situ’ colonies of cells.     

Rapid interphase analysis for prenatal diagnosis of translocation carriers using subtelomeric probes

Interphase fluorescence in situ hybridization (FISH) has become an accepted laboratory technique for the rapid and preliminary prenatal assessment of chromosome aneuploidy. The introduction of subtelomeric FISH probes now allows for the molecular–cytogenetic analysis of terminal chromosome rearrangements. In a prospective study, we examined the prenatal use of subtelomeric probes on interphase cells to rapidly detect the carrier status of a fetus when a parent carried a known reciprocal or Robertsonian chromosome translocation. Three of the cases were identified as being abnormal. All cases were confirmed by routine cytogenetic analysis. These findings clearly demonstrated the utility of this technique and these probes to rapidly and correctly identify balanced and unbalanced chromosome anomalies of a fetus that could result from parental translocations. Copyright © 2002 John Wiley & Sons, Ltd.