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.     

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.     

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 diagnosis of two rare de novo structural aberrations of the Y chromosome: cytogenetic and molecular analysis

Two rare de novo structural aberrations of the Y chromosome were detected during routine prenatal diagnosis: a satellited non-fluorescent Y chromosome (Yqs), the first de novo Yqs to be reported in a fetus, and a terminal deletion of the Y chromosome long arm del(Y)(q11). In both cases detailed cytogenetic and molecular analyses were undertaken. In the case of the Yqs it was demonstrated by fluorescence in situ hybridization (FISH) that the satellites were derived from chromosome 15. In the case of the del(Yq), it was shown with molecular analysis by polymerase chain reaction (PCR) amplification of sequence-tagged sites (STS-PCR) that the deleted portion of the long arm of chromosome Y included the azoospermia factor loci, AZFb and AZFc. The clinical significance of these findings is discussed. Copyright © 2001 John Wiley & Sons, Ltd.     

Del(18p) syndrome with increased nuchal translucency in prenatal diagnosis

We report a de novo translocation between chromosome 15 and 18 resulting in monosomy 18p in prenatal diagnosis. The patient was referred for amniocentesis due to increased nuchal translucency (INT) (5 mm) at 13.6 weeks of gestation. Karyotype of the fetus revealed 45,XX,der(15;18)(q10;q10) in all metaphases. The targeted fetal ultrasound at 20 weeks of gestation did not show any special physical abnormalities other than 6.4 mm of nuchal fold thickness. Molecular cytogenetic findings using CGH and FISH confirmed the del(18p) with dicentromeres from both chromosome 15 and 18. The present study shows that the INT at first trimester was the only prenatal finding for the fetus with del(18p) syndrome and that molecular cytogenetic methods are useful for detecting chromosomal aberrations precisely. Copyright © 2004 John Wiley & Sons, Ltd.     

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.     

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.     

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.     

Prenatal diagnosis of sacrococcygeal teratoma with constitutional partial monosomy 7q/trisomy 2p

We report the prenatal diagnosis of a fetus with sacrococcygeal teratoma and facial dysmorphism attributed to a constitutional terminal deletion of chromosome 7q and partial trisomy of chromosome 2p likely resulting from a de novo balanced translocation. The cytogenetic abnormality was diagnosed prenatally after sonographic detection of teratoma and confirmed on peripheral blood cells at birth. The newborn died of post-operative complications at seven days of age. FISH analysis demonstrated haploinsufficiency of HLXB9, a gene identified in the triad of a presacral mass (teratoma or anterior meningocele), sacral agenesis, and anorectal malformation, which constitutes the Currarino syndrome. Despite the absence of other features of the triad, the teratoma observed in the fetus we describe might represent a partial form of Currarino syndrome. Copyright © 2003 John Wiley & Sons, Ltd.     

Multicolour spectral karyotyping for complex chromosomal rearrangements in repeated abortion or congenital anomalies

Advances in molecular cytogenetics, especially the technique of fluorescence in situ hybridization (FISH), have allowed more precise definition of chromosomal structures, which are difficult to identify using conventional G-banding. Recently, a novel approach based on hybridization of 24 fluorescent-labelled chromosome painting probes was developed, termed spectral karyotyping (SKY), which allows the simultaneous and differential colour display of all human chromosomes. We have used SKY to identify not only five parental complex translocation carriers but also minute chromosome rearrangements in the fetus. Here, we concentrate attention on the clinical application of SKY for prenatal diagnosis. Copyright © 2001 John Wiley & Sons, Ltd.     

Prenatal aneuploidy detection in interphase cells by fluorescence in situ hybridization (Fish)

FISH is a quick, inexpensive, accurate, sensitive and relatively specific method for aneuploidy detection in samples of uncultured chorionic villus cells and amniotic fluid cells. FISH allows detection of the autosomal trisomies 13, 18 and 21 and X and Y abnormalities and any other chromosome abnormality for which a specific probe is available. The detection rate of these abnormalities is high in informative samples which have a concordance of > 99.5% with cytogenetic results. A relatively high number of abnormal cases are found in uninformative samples. However, such samples should be regarded as samples to be investigated further. Clinical experience with the use of FISH for prenatal diagnosis is now beyond 10,000 cases; a number of clinical protocols and smaller trials have also been carried out, resulting in 90% of attempted analyses giving informative results with a high detection rate and extraordinarily low false-positive and false-negative rates Unsolved problems remain, such as occasional technical failures, admixtures of maternal blood and up to 20% uninformative scoring results, especially for abnormal specimens. FISH is at present used as an adjunct to classical cytogenetic analysis. However, this should not be interpreted as meaning that FISH could not be used as a methodology in its own right. If FISH were to be considered a Diagnostic test then this might be the case, due to the risk of false-negative and false-positive results and the fact that FISH does not allow a diagnosis of certain structural abnormalities. If, on the other hand, FISH is considered a screening test, which means that in all abnormal (or indeterminate) cases, classical cytogenetic analysis would follow the abnormal screening test, the accuracy which is potentially higher than for other screening methods, for example in cases of trisomy 21, justifies FISH as a prenatal screening test in its own right.     

Duplication of 9 p11.2-p13.1: a benign cytogenetic variant

The detection of very rare variants in prenatal diagnosis often causes counseling difficulties and anxiety in parents. We describe a duplication of the proximal region of chromosome 9 short arm in two cases of prenatal diagnosis and in one young woman, with evidence that such rearrangement is an uncommon variant. The duplication was investigated using Fluorescence in situ hybridization (FISH). Although the cytogenetic findings were indicative of a ‘duplication 9p syndrome’ associated with mental and developmental retardation, we were able to demonstrate that the rearrangement was a heteromorphism with no phenotypic consequence. We also determined the breakpoint regions of the rearrangement and identified the BAC probes that precisely define the duplicated region devoid of risk of phenotypic effects. Copyright © 2004 John Wiley & Sons, Ltd.     

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.     

Increased fetal nuchal fold leading to prenatal diagnosis of ring chromosome 15

We report on the prenatal diagnosis of ring chromosome 15 in a fetus with increased nuchal fold and intrauterine growth restriction (IUGR). A 27-year-old woman gravida 2, para 1 had normal maternal serum screen tests in the early second trimester of the index pregnancy. Fetal nuchal fold thickening up to 8 mm was incidentally found during the routine obstetric ultrasound scan at 20 weeks' gestation. Amniocentesis was undertaken and the fetal karyotype was found to be 46,XY,r(15) on cytogenetic study. Fluorescence in situ hybridization (FISH) using a telomeric probe of chromosome 15 demonstrated a terminal deletion on the q arm of the ring-shaped chromosome 15. This is the first report of a prenatally diagnosed case of ring chromosome 15. Moreover, nuchal fold thickness in the second trimester may have a role in its prenatal diagnosis. Copyright © 2001 John Wiley & Sons, Ltd.     

A case of ring chromosome 22 with deletion of the 22q13.3 region associated with agenesis of the corpus callosum,fornix and septum pellucidum

We report a 16-week-gestation foetus obtained by voluntary abortion after prenatal diagnosis, in which a ring chromosome 22 was observed with deletion of the 22q13.3 region. A prenatal study of the amniotic fluid by standard chromosome technique with G bands and FISH (fluorescence in situ hybridisation) was performed. After the abortion, the anatomopathological study of the obtained foetus was carried out. Morphological and histological analysis of the foetus did not reveal severe physical abnormalities, although alterations of the nervous system were observed consisting of corpus callosum, fornix and septum pellucidum agenesia. It could be that the genes in this region that were involved in the development of the central nervous system were responsible for the alterations found in the morphological study. The wide range of manifestations observed in patients with this cytogenetic alteration is probably due to size differences in the deleted region. Copyright © 2004 John Wiley & Sons, Ltd.     

Fully automated FISH examination of amniotic fluid cells

Objective Fluorescence in situ hybridization (FISH) analysis has become a valuable adjunct in cytogenetics, providing a rapid screen for common chromosome abnormalities that is particularly helpful in prenatal diagnosis. FISH analysis using standard microscopy is expensive and labor intensive, requiring both a high skill level and subjective signal interpretation. A reliable fully automated system for FISH analysis could improve laboratory efficiency and potentially reduce errors and costs. Methods The efficacy of an automated system was compared to standard manual FISH analysis. Two sets of slides were generated from each of 152 amniotic fluid samples. Following hybridization with a standard panel of five chromosome FISH probes, one set of slides was evaluated using manual microscopy. The other set was evaluated using an automated microscopy system. Results A diagnostic outcome was obtained for all 152 samples using manual microscopy and for 146 of 152 (96%) samples using automated microscopy. Three cases of aneuploidy were detected. For those samples for which a diagnostic outcome was determined by both manual and automated microscopy, 100% concordance was observed. All FISH analysis results were confirmed by karyotype. Conclusion These data suggest that an automated microscopy system is capable of providing accurate and rapid enumeration of FISH signals in amniocytes. Copyright © 2007 John Wiley & Sons, Ltd.     

Preimplantation genetic diagnosis of pericentric inversions

Inversions are structural chromosome abnormalities that may be associated with infertility, multiple miscarriage and chromosomally unbalanced offspring. Preimplantation genetic diagnosis (PGD) with subtelomeric probes was used to select for transfer only those embryos that were normal or balanced for three pericentric inversions. In contrast to previous protocols the present procedure allows the detection of unbalanced embryos that might arise from U-recombination in the inverted region. Additionally, aneuploidy screening was carried out in two cases by a second round of fluorescent in situ hybridization (FISH) with centromeric probes. Of the three couples that underwent the procedure one became pregnant twice. The first pregnancy delivered a healthy and chromosomally normal baby and the second pregnancy is ongoing with triplets. Copyright © 2001 John Wiley & Sons, Ltd.     

Trisomy 15 mosaicism owing to familial reciprocal translocation t(1;15): implication for prenatal diagnosis

We describe a 4-year-old female child with severe global mental retardation, myoclonic epilepsy, proximal hypotonia and dysmorphisms, whose prenatal diagnosis following amniocentesis revealed a constitutional female karyotype carrying a t(1;15)(q10;p11) familial reciprocal translocation. Post-natal high-resolution karyotype, Fluorescence in situ hybridization (FISH) screening for subtelomeric rearrangements, VNTR search for UPD15 in the blood and fibroblast, and WCP1 and 15 in the mother, failed to provide an explanation for the complex clinical phenotype of the proband. Since the pachytene configuration of the translocated chromosomes defines a high probability of 3:1 segregation, an extensive workup was undertaken to look for a possibly cryptic mosaicism. Four percent of the cells with trisomy 15 was found in the peripheral blood lymphocytes examined by classical cytogenetic technique and interphase FISH analysis. The clinical features associated with cryptic trisomy 15 mosaicism and the problems concerning prenatal diagnosis and genetic counselling for carriers of translocations at high risk of 3:1 segregation are discussed. Copyright © 2006 John Wiley & Sons, Ltd.     

Prenatal diagnosis of primary anophthalmia with a 3q27 interstitial deletion involving SOX2

We report an interstitial deletion of chromosome 3q26-q28 in a fetus in which anophthalmia had been detected prenatally. FISH analysis, using BAC clones encompassing the SOX2 locus, showed that SOX2 gene was involved in the chromosomal breakpoint of the deletion. This case confirms that haploinsufficiency for SOX2 plays a crucial role in human eye development and emphasizes the necessity of careful chromosomal analysis, including FISH analysis of the 3q region, in case of prenatal discovery of anophthalmia. Copyright © 2004 John Wiley & Sons, Ltd.     

Fetal translocation between chromosomes 2, 18, and 21 resolved by fish

An apparently balanced t(2q;21q) translocation was discovered in fetal blood and amniocytes of a 22-week fetus, monitored because of ultrasonographic evidence of a heart disease. FISH (fluorescence in situ hybridization) analysis disclosed a complex translocation between chromosomes 2q, 18q, and 21q, which was inherited from the healthy mother. This observation corroborates the usefulness of molecular cytogenetic techniques in raising the quality of prenatal diagnosis and detecting subtle rearrangements not resolved by standard cytogenetics.