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.     

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.     

Rapid sexing of human embryos by non-radioactive in situ hybridization: Potential for preimplantation diagnosis of X-linked disorders

Sixty spare human embryos at various stages of preimplantation development were prepared for cytogenetic analysis. Fluorescent staining of those with metaphases allowed scoring for the presence of a Y chromosome. In situ hybridization was then performed using a biotinylated Y-specific sequence, and the probe was detected by a standard streptavidinlinked alkaline phosphatase system. This enabled comparison of the chromosomal sex with that obtained after in situ hybridization in 28 embryos, and the sexing result obtained by the two methods was concordant in all cases. A further 21 embryos in which no metaphase chromosomes were obtained were sexed by biotinylated in situ hybridization only. Overall, 66 per cent of male interphase nuclei demonstrated a Y-specific hybridization signal. Results were obtained in under 24 h, which may permit the sexing of an embryo biopsied during cleavage and the transfer of sexed embryos at the blastocyst stage to the mother's uterus in the same cycle as oocytes are collected for in vitro fertilization.     

Cross-hybridization of the chromosome 13/21 alpha satellite DNA probe to chromosome 22 in the prenatal screening of common chromosomal aneuploidies by fish

In a routine application of commercially available centromeric DNA probes for the prenatal screening of common trisomies involving the autosomes 13, 18, and 21, and sex chromosomes, four cases of discrepancy between fluorescence in situ hybridization (FISH) results and follow-up cytogenetic analysis were observed from a total of 516 cases of amniocentesis. In three of these cases, the results were false negative, and in one false positive. In this case, amniocentesis was performed because of a positive triple test in a 34-year-old woman with previous infertility treatment. The alpha satellite DNA probe for chromosomes 13/21 revealed five signals in 50 per cent of uncultured amniocytes, while standard cytogenetic analysis showed a normal karyotype. FISH analysis on metaphase chromosomes demonstrated the location of the additional signal in the centromeric region of chromosome 22. This additional signal was also present in the centromeric region of chromosome 22 of the mother, providing evidence for a possible inherited polymorphism in chromosome 22 responsible for unspecific hybridization with the alpha satellite probe for chromosomes 13/21 in this case. The observed polymorphism in centromeric regions may contribute to unreliability of the use of the 13/21 alpha satellite probe for prenatal screening by FISH.     

Proposed guidelines for diagnosis of chromosome mosaicism in amniocytes based on data derived from chromosome mosaicism and pseudomosaicism studies

Currently, accepted protocol which has been developed at the Prenatal Diagnosis Laboratory of New York City (PDL) requires that when a chromosome abnormality is found in one or more cells in one flask, another 20–40 cells must be examined from one or two additional flasks. Chromosome mosaicism is diagnosed only when an identical abnormality is detected in cells from two or more flasks. In a recent PDL series of 12 000 cases studied according to this protocol, we diagnosed 801 cases (6.68 per cent) of single-cell pseudomosaicism (SCPM), 126 cases (1.05 per cent) of multiple-cell pseudomosaicism (MCPM), and 24 cases (0.2 per cent) of true mosaicism. Pseudomosaicism (PM) involving a structural abnormality was a frequent finding (2/3 of SCPM and 3/5 of MCPM), with an unbalanced structural abnormality in 55 per cent of SCPM and 24 per cent of MCPM. We also reviewed all true mosaic cases (a total of 50) diagnosed in the first 22000 PDL cases. Of these 50 cases, 23 were sex chromosome mosaics and 27 had autosomal mosaicism; 48 cases had numerical abnormalities and two had structural abnormalities. Twenty-five cases of mosaicism were diagnosed in the first 20 cells from two flasks, i.e., without additional work-up, whereas the other 25 cases required extensive work-up to establish a diagnosis (12 needed additional cell counts from the initial two culture flasks; 13 required harvesting a third flask for cell analysis). Our data plus review of other available data led us to conclude that rigorous efforts to diagnose true mosaicism have little impact in many instances, and therefore are not cost-effective. On the basis of all available data, a work-up for potential mosaicism involving a sex chromosome aneuploidy or structural abnormality should have less priority than a work-up for a common viable autosomal trisomy. We recommend revised guidelines for dealing with (1) a numerical versus a structural abnormality and (2) an autosomal versus a sex chromosome numerical aneuploidy. Emphasis should be placed on autosomes known to be associated with phenotypic abnormalities. These new guidelines, which cover both flask and in situ methods, should result in more effective prenatal cytogenetic diagnosis and reduced patient anxiety.     

Preimplantation diagnosis

Research towards preimplantation diagnosis of genetic disease was initiated in the UK. in the mid 1980s with the aim of helping those couples who would prefer selection to occur at this stage rather than during pregnancy. Following in vitro fertilisation, (IVF), biopsy and removal of 1 or 2 of the totipotent cells from the cleavage stage 3 day old embryo provides the material for molecular genetic diagnosis without interfering with development. Earliest applications were in the avoidance of X-linked disease by sexing embryos and selecting females for transfer to the mother. Initially, polymerase chain reaction (PCR) amplification of DNA from the biopsied blastomeres was performed using primers specific for sequences derived from the Y chromosome and this led to the birth of several normal girls. To reduce the risk of misdiagnosis due to amplification failure, PCR based methods for sexing the embryo now employ both X and Y specific sequences, but the preferred method is currently considered to be fluorescent in situ hybridisation (FISH) with fluorochrome labelled DNA probes to the embryonic nuclei that have been fixed and spread on slides. Dual FISH with probes from X and Y chromosomes allows unequivocal diagnosis of sex and determination of chromosome copy number, avoiding transfer of embryos with abnormal numbers of sex chromosomes, including those with only the maternal X that would be at 50% risk for the X-linked disease. The application of FISH for preimplantation diagnosis has also led to the realisation that chromosomal mosaicism is common at the cleavage stage of development, a finding that has important implications for diagnosis of both dominant single gene disorders and trisomies, as well as for our understanding of early human development. Cloning and sequencing of the relevant genes has enabled the development of methods for the diagnosis of certain recessive single gene disorders in cleavage stage embryos. PCR based methods have to be developed for each condition, sometimes for each family if there is heterogeneity. Preimplantation diagnosis has been successful so far for cystic fibrosis, Tay Sachs disease, and Lesch-Nyhan syndrome. Worldwide, 32 pregnancies have been established following all types of preimplantation diagnosis and with 29 babies born, there is no evidence for any adverse effect on development.     

Prenatal detection of 45,X/46,XX/47,XXX mosaicism through amniocentesis: Mosaicism confirmed in cord blood,amnion, and chorion

Sex chromosome mosaicism in amniotic fluid cells poses a serious dilemma in prenatal diagnosis. Chromosome analysis of 56 primary clones of amniocytes revealed three distinct cell lines. Nine cells (16.1 per cent) demonstrated a 45,X karyotype, 11 cells (19.6 per cent) a 47,XXX karyotype, and the remaining 36 cells (64.3 per cent) had a modal number of 46 chromosomes (46,XX). Cytogenetic evaluation of 100 cells from cord blood, amnion, and chorion following delivery confirmed this triple mosaicism. However, the distribution of the three karyotypes in the pre- and postnatal samples was not found in the same proportions. The cord blood had the most similar frequency to that of the amniotic fluid sample, while the chorion had a significantly increased frequency of 47,XXX cells (41 per cent) and a decreased frequency of 45,X cells (2 per cent). Physical examination of the infant at birth revealed no discernible phenotypic abnormalities. Parental karyotypes were normal. This case highlights the difficulty in determining whether a prenatally detected abnormality will be associated with postnatal phenotypic deviation.     

United states survey on chromosome mosaicism and pseudomosaicism in prenatal diagnosis

The survey of the incidence of chromosome mosaicism and pseudomosaicism detected in prenatal diagnosis included data from approximately 60 000 genetic amniocenteses in the United States. There were 59 participating cytogenetic laboratories nationwide. The overall incidence of chromosome mosaicism was 0.25 per cent (range of 0–0.89 per cent). The average frequency of pseudomosaicism involving multiple cells or clones was 0.7 per cent (range of 0–11.21 per cent). The frequency of single cell or clone pseudomosaicism was 2.47 per cent (range of 0–11.49 per cent). In cases of pseudomosaicism with trisomy, the most frequently involved chromosome was number 2; occurrence rates of trisomies 7,X,9,17 and 20 were also relatively high. In cases of pseudomosaicism with structural abnormalities, this survey showed an association between relative chromosome size and the frequency of involvement in structural rearrangement. Data on a total of 185 cases of chromosome mosaicism collected in this survey as well as from other documented sources showed 89 cases involved an autosome, 13 cases a sex chromosome, and 23 a marker chromosome. The frequency of noticeable phenotypic abnormalities was highest (37.8 per cent) in the autosomal mosaics and lowest (10.5 per cent) in the sex chromosome mosaics. The average rate for cytogenetic confirmation was 70 per cent.     

Prenatal diagnosis of familial ring 21 chromosome

Ring chromosome 21 is a rare chromosome anomaly often associated with mental retardation and dysmorphic features. Less commonly, the ring chromosome can be familial and associated with a normal phenotype. Phenotypically normal female carriers, however, are at increased risk of having children with Down syndrome, mosaic monosomy 21, and variable duplication or deletion of chromosome 21. Because of the relative mitotic and meiotic instability of ring chromosomes, abnormal cytogenetic findings encountered during prenatal diagnosis may not reflect the true genetic status of the fetus. This is a report of a phenotypically normal female carrier of a familial ring 21 chromosome. Prenatal diagnosis on her twin pregnancy revealed a mosaic 46,XX,r(21)(p13;q22) (77 per cent)/45,XX, – 21 in one fetus and a normal male karyotype in the second. The pregnancy was carried to term. Both infants are completely normal, with a non-mosaic ring 21 karyotype from the lymphocytes of one twin. The diagnostic uncertainty and problematic genetic counselling related to fetal cytogenetic abnormalities are the subjects of this report.     

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.     

Cytogenetic analysis of chorionic villi for prenatal diagnosis: An ACC collaborative study of U.K. data

A prospective 3-year collaborative study was undertaken in 1987 to collect cytogenetic data from diagnostic chorionic villus samples (CVS) in the U.K. in order to determine the predictive value of the chromosome abnormalities encountered. Twenty-seven laboratories contributed a total number of 7595 cases, of which 97·6 per cent were successful. Excluding single cell anomalies, a total of 480 cytogenetic abnormalities were reported, of which 137 were familial structural rearrangements and 343 were de novo problems. Non-mosaic trisomies of chromosomes 13, 18, and 21 (n=157), non-mosaic sex chromosome abnormalities (n=33), and triploidy (n=6) were all confirmed in cells of fetal origin where follow-up information was available. Of the nine remaining non-mosaics including tetraploidy, trisomies of other autosomes, and extra markers, only a trisomy 16 and a case of a supernumerary marker proved genuine. Eighty-eight cases of mosaicism were reported to the study, of which only nine were confirmed as genuine: two cases involving chromosome 13, one trisomy 18, two examples of extra marker chromosomes, three 45,X, and one 47,XXX. There were no reports of false-negative findings. Presumptive maternal cell contamination was encountered in 39 cases, a detected incidence of 0·5 per cent. Four cases of presumptive ‘vanishing twin’ were recorded: in three of these, direct preparations showed a female karyotype, whereas cultures indicated a male (with male fetuses in two cases). The fourth case was of a female fetus with male and female cells in the CVS cultures. Subtle structural chromosome abnormalities were missed in three instances. Accurate prediction of the fetal karyotype was shown to require detailed knowledge of both the nature and the distribution of abnormal cells in the extra-embryonic tissues. In many cases, this could only be made where results from direct preparations and cultured cells were available. A number of conclusions were reached from these and similar data in the literature regarding the reliability of chromosome findings in CVS.     

Prenatal diagnosis of Pallister–Killian syndrome: Resolution of cytogenetic ambiguity by use of fluorescent in situ hybridization

We report a case of Pallister-Killian syndrome initially diagnosed prenatally as tetrasomy 21. A 33-year-old primiparous woman was noted at 24 weeks' gestation to have moderate polyhydramnios. Ultrasonography showed diminished fetal stomach filling, hydronephrosis, and prominence of the cisterna magna. Cytogenetic analysis of cultured amniocytes was initially interpreted as mosaic tetrasomy 21: 46,XX/47,XX,+i(21q). The patient was then referred to our centre for genetic counselling. At 34 weeks' gestation, a dysmorphic infant was delivered and died within 30 min. Physical features were consistent with the Pallister-Killian syndrome. Renal, gastrointestinal, and central nervous system anomalies were found at post-mortem examination. Analysis of peripheral lymphocytes revealed 5 per cent of cells with a marker chromosome, while 92 per cent of cultured fibroblasts had this same marker. Fluorescent in situ hybridization (FISH) using an alpha-satellite probe for chromosomes 13 and 21 failed to hybridize to the marker, while a chromosome 12 centromeric probe unequivocally identified it as an i(12p). Use of FISH can provide rapid, specific prenatal diagnosis of ambiguous marker chromosomes and improve prenatal counselling.     

Discordance between prenatal cytogenetic diagnosis and outcome of pregnancy

From 1.3.73 to 30.9.80 5580 women had an amniocentesis performed here or elsewhere; fetal chromosome analyses were carried out in this laboratory. We found 112 abnormal karyotypes (2 per cent) out of 5591 chromosome analyses. In 40 women (0.7 per cent) no cytogenetic diagnosis was obtained. Follow-up was successful in 99.5 per cent. Nine cases are reported in detail: Three cases had discrepancy between the karyotype in amniotic fluid and peripheral blood after delivery, two of these cases turned out to be 46,XX (male) while the third was prenatally determined as trisomy 21, but had a 46,XX karyotype at birth. Six cases had discrepancy between the karyotype in amniotic fluid and the phenotypic outcome at birth/abortion. One case was a prenatally undetected 45,X/46,XY mosaicism; one case was an unexplained 45,X male fetus; two cases were prenatally determined as trisomy 21, but at abortion a normal karyotype was determined and in two cases maternal cells were probably examined. The incidence of cytogeneric errors in this study was very low.     

Chromosome analysis of multipronuclear human oocytes after in vitro fertilization

Multipronuclear human eggs are frequent after in vitro fertilization. Their chromosome analysis can provide useful information. Before cleavage it can confirm the suspected poly-ploidy. Among the cleaved multipronuclear eggs it provides an estimation of the incidence of the possible return to diploidy. Ninety-four multipronuclear eggs were fixed at the first, second, or third cleavage according to the air-drying method of Tarkowski with or without colchicine exposure: 60 were successfully analysed. Twelve were stopped before cleavage (six without colchicine treatment and six with colchicine treatment). They were polyploid, confirming the cytological observation. Forty-eight eggs cleaved and were stopped by colchicine treatment and karyotyped. Seventeen eggs (35 per cent) had produced diploid embryos. Mosaicism was frequent (15 cases, 31 per cent). Triploidy was not frequent (8 eggs, 17 per cent). Haploidy constituted the remaining cases (8 eggs, 17 per cent). Our data indicate that the initial count of pronuclei is a reliable test. Multipronuclear one-cell oocytes were confirmed to be polyploid. Furthermore, the developmental capacity of the multipronuclear oocytes is variable. Most of them cleaved. However, many multipronuclear oocytes led to diploid cleaving eggs.     

A canadian collaborative study of mosaicism in amniotic fluid cell cultures

Data on chromosomal mosaicism was collected retrospectively on 12 386 amniotic fluid samples cultured over a 10 year period in 14 Canadian centres. Level I mosaicism (a single abnormal cell—excluding single cell monosomy) was encountered in 863 cases (7.1 per cent). Level II mosaicism (multiple cells with the same abnormality in a single flask or colony) was encountered in 138 cases (1.1 per cent). Level III mosaicism (multiple cells distributed over multiple flasks or colonies) was encountered in 34 cases (0.3 per cent). Analysis of the details of these cases allowed five major conclusions to be drawn: (1) Single cell abnormalities should not be taken as an indication of true fetal mosaicism. Only rarely will this interpretation prove to be incorrect. (2) Mosaicism involving multiple cells confined to a single flask should not be regarded as an indication of true fetal mosaicism. Only occasionally will this interpretation prove to be incorrect. (3) Mosaicism involving multiple cells distributed over more than one flask should be regarded as a strong indication of true fetal mosaicism. Sixty per cent will be confirmed by karyotype analysis of the fetus or infant. (4) Mosaicism of the XX/XY type is usually due to maternal cell contamination. Occasionally it can be a female fetus with XY cells from an unknown source. (5) The in situ or colony method of chromosome analysis has no clear advantage over the flask method for either the detection of true fetal mosaicism or for the ability to distinguish true mosaics from false positives.     

Prenatal diagnosis of ring chromosome 6

An amniocentesis was performed on a gravida 1, para 0 23-year-old female because of high maternal serum alpha-fetoprotein and nuchal thickening/cystic mass apparent on the fetal ultrasound. Detailed ultrasound examination revealed multiple anomalies including brain abnormalities. The fetus was found to have a mosaic female karyotype: 45,XX, - 6/46,XX,r(6) (p25q27) (62 per cent:38 per cent). This is the first report of a prenatally diagnosed case of ring chromosome 6.     

Is placental mosaicism associated with poor perinatal outcome?

In 37 pregnancies with placental mosaicism detected at first-trimester chorionic villus sampling in 2000 pregnancies, two pregnancies (5.4 per cent) ended in a spontaneous abortion at 15 weeks (46,XX/47,XX, + 16) and in a perinatal loss in a term pregnancy (46,XY/ 47,XY, +10), respectively. The outcome of the other 35 pregnancies was normal. The total pregnancy loss rate before 28 weeks in the first 1000 pregnancies was previously reported to be 3.6 per cent.     

Mosaicism or pseudomosaicism: The problem of hypermodal cells in amniotic fluid cell culture

A series of 2029 consecutive amniotic fluid specimens studied for prenatal genetic diagnosis were reviewed and reassessed so as to evaluate the frequency and clinical significance of hypermodal cells in amniotic fluid cell cultures. Hypermodal cells were defined as those with more than 46 chromosomes, and were characterized by an additional structurally normal or structurally abnormal chromosome. Of 2029 specimens, 47 (2.31 per cent) contained a total of 167 hypermodal cells. True fetal mosaicism was detected in three cases (0.14 per cent). All had hypermodal cells in more than one culture flask or colony which contained the same aberrant chromosome complement. In all but one case the babies were normal when only one cell was hypermodal, or when several cells were hypermodal but present in only one colony or one culture vessel. One case had an extra No. 20 chromosome in one cell. Although the child had multiple anomalies, they were not characteristic of trisomy 20, and subsequent chromosomal study on the baby postnatally revealed a 46,XX karyotype. The in situ coverslip technique is recommended as the preferred method for prenatal diagnosis, and it is useful as an aid in differentiating true mosaicism from pseudomosaicism.     

Characterization of marker chromosomes by microdissection and fluorescence in situ hybridization

We characterized by microdissection and fluorescence in situ hybridization (FISH) two marker chromosomes: (1) a de novo, acrocentric marker chromosome detected in 88 per cent of the amniotic fluid cells of one of two physically and developmentally normal twins; and (2) a metacentric marker chromosome present in a phenotypically normal female. Analysis of FISH probes developed from the marker chromosomes indicated that the marker chromosomes in cases 1 and 2 were del(14)(q11) and a derivative chromosome from a Robertsonian translocation, respectively. Microdissection in combination with FISH may prove to be a valuable technique in determining the chromosomal origin of de novo marker chromosomes and unbalanced structural rearrangements detected during prenatal diagnosis.     

Prenatal diagnosis of a small supernumerary,XIST-negative,mosaic ring X chromosome identified by fluorescence in situ hybridization in an abnormal male fetus

Marker or ring X [r(X)] chromosomes of varying size are often found in patients with Turner syndrome. Patients with very small r(X) chromosomes that did not include the X-inactivation locus (XIST) have been described with a more severe phenotype. Small r(X) chromosomes are rare in males and there are only five previous reports of such cases. We report the identification of a small supernumerary X chromosome in an abnormal male fetus. Cytogenetic analysis from chorionic villus sampling was performed because of fetal nuchal translucency thickness and it showed mosaicism 46,XY/47,XY,+r(X)/48,XY,+r(X),+r(X). Fluorescence in situ hybridizations (FISH) showed the marker to be of X-chromosome origin and not to contain the XIST locus. Additional specific probes showed that the r(X) included a euchromatic region in proximal Xq. At 20 weeks gestation, a second ultrasound examination revealed cerebral abnormalities. After genetic counselling, the pregnancy was terminated. The fetus we describe is the first male with a mosaic XIST-negative r(X) chromosome identified at prenatal diagnosis. The phenotype we observed was probably the result of functional disomy of the genes in the r(X) chromosome, secondary to loss of the XIST locus. Copyright © 2003 John Wiley & Sons, Ltd.