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.     

A 10-year survey, 1980–1990, of prenatally diagnosed small supernumerary marker chromosomes,identified by fish analysis. Outcome and follow-up of 14 cases diagnosed in a series of 12 699 prenatal samples

A cytogenetic survey and follow-up studies were made of 14 cases with supernumerary marker chromosomes, identified among 12 699 prenatal samples, investigated at our institution over a 10-year period from 1980 to 1990. FISH (fluorescence in situ hybridization) techniques were employed to identify the chromosomal origin of the marker chromosomes. Five cases were familial, all derived from acrocentric chromosomes, and all without apparent phenotypic effects in the children. Nine cases represented de novo aberrations. In two cases (one with a marker from chromosome 14 or 22, the other with a ring-like marker derived from chromosome 17), the pregnancies continued and apparently normal babies were delivered at term, but the child with a marker derived from chromosome 17 showed slight psychomotor retardation at 2 years of age. All other pregnancies with de novo markers were terminated. In three cases, significant abnormalities were found at autopsy. One of these had an isochromosome 12p and the phenotype was consistent with Pallister-Killian syndrome. In conclusion, marker chromosome identification, as well as clinical follow-up, is essential for the purpose of improving genetic counselling.     

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 identification of an isochromosome for the short arm of the Y i(Yp), by cytogenetic and Molecular analyses

A case of 45,X/46,X,+mar mosaicism was detected in a male fetus (27 weeks' gestation) referred for karyotype analysis following the observation of a short femur at the ultrasound scan. Analysis of 12 Y-chromosome loci by fluorescent in situ hybridization (FISH) and polymerase chain reaction (PCR) demonstrated that the marker chromosome is of Y origin and corresponds to an authentic isochromosome for the short arm of the Y chromosome, i(Yp). The breakpoint on this marker is in YQ11·1 close to the centromere. The present report illustrates the importance of FISH and PCR techniques as a complement to cytogenetic methods for accurate identification and characterization of chromosome rearrangements in prenatal diagnosis.     

Prenatal identification of i(YP) by molecular cytogenetic analysis

An i(Yp) is a rare marker chromosome. We present a case of de novo 46,X,i(Yp) detected prenatally in an amniotic fluid specimen. Fluorescence in situ hybridization (FISH) studies using a panel of Y-specific biotinylated DNA probes identified the marker chromosome as i(Yp). Comparative genomic hybridization (CGH) studies further confirmed the diagnosis. Upon pregnancy termination, external examination of the fetus revealed a generally well-developed male fetus with slight facial dysmorphism and prominent rocker-bottom feet. The molecular cytogenetic data in this case proved very useful in genetic counselling and served as a good example illustrating the important role of molecular techniques for accurate identification of marker chromosomes.     

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.     

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.     

Application of fluorescent in situ hybridization for ‘de novo’ anomalies in prenatal diagnosis

Fluorescent in situ Hybridization (FISH) was carried out for three cases of abnormal karyotypes in prenatal studies. Two concerned de novo structural anomalies and the third a marker chromosome. The origin of the extra material could be defined in all three cases, which gives a better insight into the relationship between genotype and phenotype and makes more adequate genetic counselling possible.     

Uncommon chromosomal mosaicism in chorionic villi

Three cases of unusual chromosomal mosaicism are reported for which the cytogenetic data show inconsistent findings between CVS and AC or fetal tissue, and which cannot be explained simply by non-disjunction. For case 1, in CVS the karyotype was 46,XY, whereas lymphocytes and fibroblasts revealed 69,XXY. DNA fingerprinting indicated one paternal and two maternal chromosome sets, the latter most probably due to omission of maternal meiosis II. For case 2, in CVS mos 46,XX/47,XX,+mar de novo was observed. Amniotic fluid cells had the karyotype 46,XX. The origin of the marker chromosome might be explained by at least two events of unknown order (a somatic chromosome/chromatid deletion and non-disjunction of the homologous chromosome). In case 3 (CVS: mos 46,XY/46,XY,19q+ de novo; amniotic fluid cells, lymphocytes, and fibroblasts: 46,XY), the surplus of chromosome material in 19q+ might be explained on the basis of a somatic translocation. The idea of a chimera is less convincing, as the mosaic finding is restricted to one tissue. Furthermore, there was no hint of a vanishing twin. Hitherto, no case of structural chromosome mosaicism in CVS has been reconfirmed in fetal tissues.     

Molecular and cytogenetic characterization of extra-structurally abnormal chromosomes (ESACs) found prenatally: outcome and follow-up

A 40-year-old woman underwent amniocentesis at 15.3 weeks of gestation. Chromosome analysis performed using QFQ, DA-DAPI and CBG banding revealed two de novo extra-chromosomal markers (ESACs) in 11 of the 16 colonies analysed. Fluorescence in situ hybridization (FISH) showed that both chromosomes came from the Yq11.22.1 region of the Y chromosome. PCR analysis of genes and STS localized on the Y chromosome excluded the Yp presence specifically of the SRY gene, and most of the euchromatic region of Yq. After extensive genetic counselling and considering both laboratory and second-level ultrasound data, the couple decided to continue the pregnancy. At 37.4 weeks of gestational age, a girl weighing 2750 g was born with an Apgar score of 9/10. A blood sample taken from the umbilical cord showed three cellular lines:mos47,XX, +mar1 ish.der (Y)(wcpY+) [21%]/48,XX, +mar1 ish.der (Y)(wcpY+), +mar2 ish.der (Y)(wcpY+) [41%]/46,XX [38%]. One year after birth, the baby was developing normally and had normal psychomotorial activity. Copyright © 2003 John Wiley & Sons, Ltd.     

Maternal uniparental isodisomy 10 and mosaicism for an additional marker chromosome derived from the paternal chromosome 10 in a fetus

An Erratum has been published for this article in Prenatal Diagnosis 22(11) 2002: 1056. We report a case of maternal isodisomy 10 combined with mosaic partial trisomy 10 (p12.31-q11.1). Chromosome examinations from a CVS sample showed a karyotype 47,XY,+mar/46,XY. The additional marker chromosome which was present in 6/25 interphase nuclei was shown by fluorescence in situ hybridization (FISH) to have been derived from a pericentromeric segment of chromosome 10. DNA analysis was performed from umbilical cord blood from the fetus after termination of the pregnancy at 18 weeks. The results showed that the two structurally normal chromosomes 10 were both of maternal origin, whereas the marker chromosome derived from the father. Autopsy of the fetus revealed hypoplasia of heart, liver, kidneys and suprarenal glands, but, apart from a right bifid ureter, no structural organ abnormalities. This fetus represents the second reported instance of a maternal uniparental disomy (UPD) 10. Copyright © 2002 John Wiley & Sons, Ltd.     

Mosaicism of autosomes and sex chromosomes in morphologically normal,monospermic preimplantation human embryos

We have previously detected chromosome abnormalities in human embryos whilst identifying the sex for preimplantation diagnosis of X-linked disease. In this study we assess the incidence of these abnormalities, both for sex chromosomes and autosomes 1 and 17, using dual fluorescent in situ hybridization (FISH). Sixty-nine normally fertilized embryos of good morphology at the 6–10 cell stage (day 3 post-insemination) were examined. The embryos were spread whole using HCl and Tween 20 to dissolve the cytoplasm. Thirty-four embryos were analysed for the sex chromosomes and 35 for autosomes 1 and 17. All probes were directly labelled with fluorochromes allowing analysis in 2 h. Control lymphocytes demonstrated that the probes were of high specificity. For the sex chromosomes, five embryos were mosaic (15 per cent) with the remaining 29 being uniformly XX or XY. In no case was an XX nucleus found in an otherwise XY embryo, indicating that even though mosaicism for the sex chromosomes is present, such abnormalities would not lead to a misdiagnosis of sex. For the autosomes, 16 embryos were abnormal (46 per cent); one embryo was triploid, one was monosomic for chromosome 1, and ten others were diploid mosaics (three diploid/aneuploid, three diploid/polyploid, and four diploid/haploid). A further four embryos had variable chromosome numbers in the majority of nuclei which appeared to be the result of uncontrolled mitotic division. The presence of haploidy or double monosomy, which occurred in 15 per cent of nuclei, has important implications for the diagnosis of trisomies and dominant disorders.     

Prenatal identification of small mosaic markers of different chromosomal origins

In situ hybridization using a series of alphoid DNA probes has demonstrated the origin of two small accessory mosaic marker chromosomes ascertained from 1079 amniocenteses. These markers appeared to be de novo, derived from acrocentric chromosomes, and identical by traditional cytogenetic staining (G, Q, C, AgNOR, Hoechst-distamycin). Molecular characterization showed that one marker had originated from chromosome 14, the other from chromosome 22. Clinical outcome in both cases was normal.     

Prenatal diagnosis of a double bisatellited marker with an unusual copy number ratio

Prenatal diagnosis, by amniocentesis, revealed mosaicism with respect to a bisatellited, apparently dicentric, DA/DAPI positive, de novo marker. The following cell lines were observed in decreasing order of frequency: 46,XX > 48.XX, + mar, + mar » 47,XX, + mar. The pregnancy was terminated and post-mortem examination revealed an apparently normal fetus. Cytogenetic studies of fetal and placental tissues revealed approximately the same level of mosaicism together with the unusual copy number ratio seen in the amniotic fluid cultures. Non-disjunction at the first post-zygotic mitotic division giving rise to a mosaic: 46,XX/ 48,XX, + mar, + mar followed by subsequent mitotic instability of the marker could account for the unusual copy number ratio.     

Fetoplacental discrepancy involving structural abnormalities of chromosome 8 detected by prenatal diagnosis

We describe the finding of three cell lines involving different structural abnormalities of chromosome 8 detected in a prenatal diagnosis. Chorionic villi sampling (CVS) was performed on a pregnant woman because of advanced maternal age. Semidirect cytogenetic analysis showed a mos46,XX,i(8q)/46,XX,del(8)(p11.2) karyotype, confirmed by fluorescence in situ hybridization (FISH). Amniocentesis was subsequently performed, and the karyotype obtained was 46,XX,dup(8)(p23p11.2). The pregnancy was terminated; pathologic findings included clubfeet, clenched left hand, subcutaneous edema and bilateral hydrocephalus. Molecular studies using chromosome 8 microsatellites performed on parents' blood and fetal tissues revealed a maternal meiotic origin of the inv dup(8p) with deletion of the distal p23 region and duplication of the remaining 8p. We propose a model to explain the cytogenetic findings, which includes a first maternal meiotic error giving rise to a large dicentric isochromosome 8 present in the ovum, a second error in one of the first zygote divisions with misdivision of the dicentric 8 giving rise to a cell line with del(8p) confined to the trophoblast and another cell line with inv dup(8p) confined to the fetal tissue and a third error in the trophoblast giving rise to a further cell line with isochromosome 8q. Copyright © 2003 John Wiley & Sons, Ltd.     

Prenatal diagnosis of partial tetrasomy 14: a case study

Prenatal specimens were received from a fetus with abnormalities noted on ultrasound. A supernumerary marker chromosome (SMC) was detected: 47,XY,+mar. Fluorescence in situ hybridisation (FISH) further classified this to be partial tetrasomy for chromosome 14. We compare this finding with other cases of SMC (14) and further classify phenotype with karyotype. Copyright © 2002 John Wiley & Sons, Ltd.     

Retrospective study of trisomy 18 in chorionic villi with fluorescent in situ hybridization on archival direct preparations

Trisomy 18 in direct chorionic villus preparations needs further investigation since the chromosome abnormality may be confined to the placenta and may not represent the actual fetal karyotype. We performed, retrospectively, fluorescent in situ hybridization (FISH) with the chromosome 18 centromere probe (L1.84) on interphase nuclei of destained slides of all cases of full trisomy 18 (n=22) and mosaic trisomy 18 (n=8) detected among 7600 first-trimester chorionic villus samples during an 8-year period (1985–1992). More nuclei displaying three signals were encountered in cases of full and mosaic trisomy 18 confirmed in fetal tissue than in non-confirmed cases. FISH can be useful for the verification of trisomy 18 in direct chorionic villus preparations.     

The vanishing twin: An explanation for discordance between chorionic villus karyotype and fetal phenotype

One ‘erroneous’ diagnosis occurred in 200 first-trimester chorionic villus samples (CVS) analysed. In direct preparations following 24 h incubation as well as in long-term cultures, a 46.XX karyotype was observed in the villi (28 and 25 cells, respectively). At 20 weeks of gestation, labour was induced because of fetal death in utero. An autopsy performed on the fetus revealed a male phenotype. Placenta and fetal tissues were not submitted for cytogenetic studies. The discordant CVS karyotype (46,XX), in view of the male fetal phenotype, prompted further cytogenetic and molecular studies. Chromosome marker studies on the parents' blood and chorionic villi confirmed both maternal and paternal inheritance of chromosomes in the CVS. DNA studies on formalin-fixed skin using a Y-specific probe, DYZ1, confirmed the presence of a Y chromosome in the fetus. The most likely cause of the discrepant CVS karyotype is the presence of an undetected degenerating dizygotic twin.     

Prenatal investigation of a 45,X/46,X,r(?) karyotype in amniocytes using fluorescence in situ hybridization with an X-centromeric probe

The karyotype of cultured amniotic fluid cells obtained on the indication of advanced maternal age was shown to be a mosaic 45,X/46,X,r(?). The small size and banding pattern made it difficult to determine whether the ring was derived from and X or a Y chromosome, or even from an autosome. By using an X-centromeric probe and fluorescence in situ hybridization (FISH), we demonstrated the ring to have an X centromere. Thus, a more complete genetic counselling was possible. This confirms the usefulness of FISH in identifying and characterizing this and other chromosome rearrangements in prenatal diagnosis.