Prenatal detection of aneuploidies using fluorescencein situ hybridization: A preliminary experience in an Indian set up

Fluorescencein situ hybridization (FISH) is a powerful molecular cytogenetic technique which allows rapid detection of aneuploidies on interphase cells and metaphase spreads. The aim of the present study was to evaluate FISH as a tool in prenatal diagnosis of aneuploidies in high risk pregnancies in an Indian set up. Prenatal diagnosis was carried out in 88 high-risk pregnancies using FISH and cytogenetic analysis. Multicolour commercially available FISH probes specific for chromosomes 13, 18, 21, X and Y were used. Interphase FISH was done on uncultured cells from chorionic villus and amniotic fluid samples. FISH on metaphase spreads was done from cord blood samples. The results of FISH were in conformity with the results of cytogenetic analysis in all the normal and aneuploid cases except in one case of structural chromosomal abnormality. The hybridization efficiency of the 5 probes used for the detection of aneuploidies was 100%. Using these probes FISH assay yielded discrete differences in the signal profiles between cytogenetically normal and abnormal samples. The overall mean interphase disomic signal patterns of chromosomes 13, 18, 21, X and Y were 94.45%; for interphase trisomic signal pattern of chromosome 21 was 97.3%. Interphase FISH is very useful in urgent high risk cases. The use of FISH overcomes the difficulties of conventional banding on metaphase spreads and reduces the time of reporting. However, with the limited number of probes used, the conventional cytogenetic analysis serves as a gold standard at present. It should be employed as an adjunctive tool to conventional cytogenetics     

Rapid-prenatal diagnosis through fluorescence in situ hybridization for preventing aneuploidy related birth defects

BACKGROUND AND OBJECTIVE:

Women with high-risk pregnancies are offered prenatal diagnosis through amniocentesis for cytogenetic analysis of fetal cells. The aim of this study was to evaluate the effectiveness of the rapid fluorescence in situ hybridization (FISH) technique for detecting numerical aberrations of chromosomes 13, 21, 18, X and Y in high-risk pregnancies in an Indian scenario.

MATERIALS AND METHODS:

A total of 163 samples were received for a FISH and/or a full karyotype for prenatal diagnosis from high-risk pregnancies. In 116 samples both conventional culture techniques for getting karyotype through G-banding techniques were applied in conjunction to FISH test using the AneuVysion kit (Abbott Molecular, Inc.), following standard recommended protocol to compare the both the techniques in our setup.

RESULTS:

Out of 116 patients, we got 96 normal for the five major chromosome abnormality and seven patients were found to be abnormal (04 trisomy 21, 02 monosomy X, and 01 trisomy 13) and all the FISH results correlated with conventional cytogenetics. To summarize the results of total 163 patients for the major chromosomal abnormalities analyzed by both/or cytogenetics and FISH there were 140 (86%) normal, 9 (6%) cases were abnormal and another 4 (2.5%) cases were suspicious mosaic and 10 (6%) cases of culture failure. The diagnostic detection rate with FISH in 116 patients was 97.5%. There were no false-positive and false-negative autosomal or sex chromosomal results, within our established criteria for reporting FISH signals.

CONCLUSION:

Rapid FISH is a reliable and prompt method for detecting numerical chromosomal aberrations and has now been implemented as a routine diagnostic procedure for detection of fetal aneuploidy in India.     

Characterization of a chromosomal rearrangement responsible for producing "apparent" XY-female fall-run Chinook salmon in California

Fluorescence in situ hybridization (FISH) was used to identify the X and Y chromosomes of offspring produced by normal and "apparent" XY-female fall-run Chinook salmon (Oncorhynchus tshawytscha) from California. FISH experiments were performed using probes to 2 sex-linked loci, growth hormone pseudogene (GH-Psi), and OtY1, as well as a probe to a sex-linked microsatellite (Omy7INRA). Comparison of FISH staining patterns between the offspring produced by normal and apparent XY-females revealed that the apparent XY-female examined transmitted a "Y-like" chromosome with an attenuated OtY1 and GH-Psi signal to half of its offspring. Segregation analysis of microsatellites derived from rainbow trout (Oncorhynchus mykiss) with respect to phenotypic sex was carried out for 2 normal and 2 apparent XY-female Chinook salmon families. Inheritance patterns of Omy7INRA were consistent with this locus being closely linked to GH-Psi in males and in apparent XY-females carrying the Y-like chromosome. Another microsatellite locus (Omm1077) was closely linked to the primary sex-determining locus (SEX) in males but not to GH-Psi/OtY1 in apparent XY-females. The FISH analyses suggest that apparent XY-female fall-run Chinook salmon in California are not the product of a Y chromosome to autosome translocation. Despite the combined FISH and inheritance analyses, we were unable to differentiate between 2 alternative explanations for apparent XY-females, namely, recombination of markers between the sex chromosomes, or a Y chromosome with a dysfunctional or missing sex-determining region.     

X/Y translocation in a family with Leri-Weill dyschondrosteosis

An X/Y translocation associated with Leri-Weill dyschondrosteosis (LWD) was detected in a boy and in his mother. FISH analysis with specific probes for SHOX and SRY displayed no signal on the der(X), while one signal for SHOX was detected on the normal X chromosome in the mother, and one signal each for SHOX and SRY was detected on the normal Y chromosome in the proband.     

Rapid prenatal diagnosis of chromosomal aneuploidies by fluorescence in situ hybridization: clinical experience with 4,500 specimens.

Detection of chromosome aneuploidies in uncultured amniocytes is possible using fluorescence in situ hybridization (FISH). We herein describe the results of the first clinical program which utilized FISH for the rapid detection of chromosome aneuploidies in uncultured amniocytes. FISH was performed on physician request, as an adjunct to cytogenetics in 4,500 patients. Region-specific DNA probes to chromosomes 13, 18, 21, X, and Y were used to determine ploidy by analysis of signal number in hybridized nuclei. A sample was considered to be euploid when all autosomal probes generated two hybridization signals and when a normal sex chromosome pattern was observed in greater than or equal to 80% of hybridized nuclei. A sample was considered to be aneuploid when greater than or equal to 70% of hybridized nuclei displayed the same abnormal hybridization pattern for a specific probe. Of the attempted analyses, 90.2% met these criteria and were reported as informative to referring physicians within 2 d of receipt. Based on these reporting parameters, the overall detection rate for aneuploidies was 73.3% (107/146), with an accuracy of informative results for aneuploidies of 93.9% (107/114). Compared to cytogenetics, the accuracy of all informative FISH results, euploid and aneuploid, was 99.8%, and the specificity was 99.9%. In those pregnancies where fetal abnormalities had been observed by ultrasound, referring physicians requested FISH plus cytogenetics at a significantly higher rate than they requested cytogenetics alone. The current prenatal FISH protocol is not designed to detect all chromosome abnormalities and should only be utilized as an adjunctive test to cytogenetics. This experience demonstrates that FISH can provide a rapid and accurate clinical method for prenatal identification of chromosome aneuploidies.     

Utility of Interphase FISH Panels for Routine Clinical Cytogenetic Evaluation of Chronic Lymphocytic Leukemia and Multiple Myeloma

Specific genetic abnormalities are of prognostic significance for patients with chronic lymphocytic leukemia (CLL) and multiple myeloma (MM); however, routine cytogenetic analysis usually provides normal results. We utilized two probe panels for interphase fluorescence in situ hybridization (FISH) studies to enhance the ability to detect genetic abnormalities in samples that were referred for routine cytogenetic studies for possible diagnoses of CLL or MM. The CLL panel consisted of probes for 11q22.3 (ATM gene), 13q14 (D13S319), the centromere of chromosome 12 (D12Z3) and 17p13.1 (P53 gene). The MM panel included probes for 14q32 (IgH gene) and/or t(11:14)(q13;q32) (BCL1/IgH), 13q14 (D13S319) and 17p13.1 (P53 gene). FISH detected clonal aberrations not identified by conventional cytogenetics in an additional 8 of 23 (35%) samples referred for possible CLL and 7 of 42 (17%) samples with possible MM. The prognostic significance of the aberrations identified ranged from favorable, to intermediate, to poor. Our studies indicate that many samples referred for routine cytogenetics testing for CLL and MM yield normal results for both conventional and FISH testing, likely due to lack of definitive diagnosis in a percentage of cases. However, FISH is more sensitive for the detection of clinically significant chromosome abnormalities and should be the testing methodology of choice for these disorders.     

Image cytometry-based detection of aneuploidy by fluorescence in situ hybridization in suspension

Cytogenetic abnormalities are important diagnostic and prognostic criteria for hematologic malignancies. Karyotyping and fluorescence in situ hybridization (FISH) are the conventional methods by which these abnormalities are detected. The sensitivity of these microscopy-based methods is limited by the abundance of the abnormal cells in the samples and therefore these analyses are commonly not applicable to minimal residual disease (MRD) stages. A flow cytometry-based imaging approach was developed to detect chromosomal abnormalities following FISH in suspension (FISH-IS), which enables the automated analysis of several log-magnitude higher number of cells compared with the microscopy-based approaches. This study demonstrates the applicability of FISH-IS for detecting numerical chromosome aberrations, establishes accuracy, and sensitivity of detection compared with conventional FISH, and feasibility to study procured clinical samples of acute myeloid leukemia (AML). Male and female healthy donor peripheral blood mononuclear cells hybridized with combinations of chromosome enumeration probes (CEP) 8, X, and Y served as models for disomy, monosomy, and trisomy. The sensitivity of detection of monosomies and trisomies amongst 20,000 analyzed cells was determined to be 1% with a high level of precision. A high correlation (R(2) = 0.99) with conventional FISH analysis was found based on the parallel analysis of diagnostic samples procured from 10 AML patients with trisomy 8 (+8). Additionally, FISH-IS analysis of samples procured at the time of clinical remission demonstrated the presence of residual +8 cells indicating that this approach may be used to detect MRD and associated chromosomal defects. ? 2012 International Society for Advancement of Cytometry.     

Structural unbalanced chromosome rearrangements resolved by comparative genomic hybridization.

The identification of unbalanced structural chromosome rearrangements using conventional cytogenetic techniques depends on recognition of the unknown material from its banding pattern. Even with optimally banded chromosomes, when large chromosome segments are involved, cytogeneticists may not always be able to determine the origin of extrachromosomal material and supernumerary chromosomes. We report here on the application of comparative genomic hybridization (CGH), a new molecular-cytogenetic assay capable of detecting chromosomal gains and losses, to six clinical samples suspected of harboring unbalanced structural chromosome abnormalities. CGH provided essential information on the nature of the unbalanced aberration investigated in five of the six samples. This approach has proved its ability to resolve complex karyotypes and to provide information when metaphase chromosomes are not available. In cases where metaphase chromosome spreads were available, confirmation of CGH results was easily obtained by fluorescence in situ hybridization (FISH) using specific probes. Thus the combined use of CGH and FISH provided an efficient method for resolving the origin of aberrant chromosomal material unidentified by conventional cytogenetic analysis.     

A novel 5q11.2 deletion detected by microarray comparative genomic hybridisation in a child referred as a case of suspected 22q11 deletion syndrome

The 22q11 deletion syndrome (22q11DS) is a developmental syndrome comprising of heart, palate, thymus and parathyroid glands defects. Individuals with 22q11DS usually carry a 1.5- to 3-Mb heterozygous deletion on chromosome 22q11.2. However, there are many patients with features of 22q11DS without a known cause from conventional karyotype and FISH analysis. Six patients with features of 22q11DS, a normal chromosomal and FISH 22q11 analysis, were selected for investigation by microarray genomic comparative hybridisation (array CGH). Array-CGH is a powerful technology enabling detection of submicroscopic chromosome duplications and deletions by comparing a differentially labelled test sample to a control. The samples are co-hybridised to a microarray containing genomic clones and the resulting ratio of fluorescence intensities on each array element is proportional to the DNA copy number difference. No chromosomal changes were detected by hybridisation to a high resolution array representing chromosome 22q. However, one patient was found to have a 6-Mb deletion on 5q11.2 detected by a whole genome 1-Mb array. This deletion was confirmed with fluorescence in-situ hybridisation (FISH) and microsatellite marker analysis. It is the first deletion described in this region. The patient had tetralogy of Fallot, a bifid uvula and velopharyngeal insufficiency, short stature, learning and behavioural difficulties. This case shows the increased sensitivity of array CGH over detailed karyotype analysis for detection of chromosomal changes. It is anticipated that array CGH will improve the clinicians capacity to diagnose congenital syndromes with an unknown aetiology.     

Specific cytogenetic labeling of bovine spermatozoa bearing X or Y chromosomes using fluorescent in situ hybridization (FISH)

X and Y specific probes were identified in order to apply the fluorescent in situ hybridization (FISH) technique to bovine spermatozoa. For Y chromosome detection, the BRY4a repetitive probe, covering three quarters of the chromosome, was used. For X chromosome detection, a goat Bacterial Artificial Chromosome (BAC) specific to the X chromosome of bovine and goats and giving a strong FISH signal was used. Each probe labeled roughly 45% of sperm cells. The hybridization method will be useful for evaluating the ratio of X- and Y- bearing spermatozoa in a sperm sample and consequently can be used to evaluate the efficiency of sperm sorting by different techniques such as flow cytometry.     

Different chromosome Y abnormalities in Turner syndrome.

A 17-year-old phenotypically female girl was referred for evaluation because of short stature and primary amenorrhea. Cytogenetic analysis showed a mosaic 46,XY/45,X/47,XYY/46,X,idic(Yq)/47,XY,idic(Yq)/48,XXY,idic(Yq)/46,X,t(C;Y) karyotype. Conventional cytogenetic results were supplemented with fluorescence in situ hybridization (FISH) techniques to ensure a better characterization of abnormalities. By using FISH, a supernumerary marker chromosome derived from chromosome Y which could not be detected by conventional cytogenetics was revealed. Furthermore, additional abnormalities and their frequencies were highlighted by the application of DNA probes specific for X and Y chromosomes. Thus, FISH proved useful in determining low frequency cell lines which would need analysis of a large number of good quality metaphase spreads by conventional cytogenetic techniques: it helped in identifying the nature and the origin of unknown markers and rearrangements which have important implication in sexual differentiation and development of gonadal tumours.     

Comparative genomic hybridization in clinical cytogenetics.

We report the results of applying comparative genomic hybridization (CGH) in a cytogenetic service laboratory for (1) determination of the origin of extra and missing chromosomal material in intricate cases of unbalanced aberrations and (2) detection of common prenatal numerical chromosome aberrations. A total of 11 fetal samples were analyzed. Seven cases of complex unbalanced aberrations that could not be identified reliably by conventional cytogenetics were successfully resolved by CGH analysis. CGH results were validated by using FISH with chromosome-specific probes. Four cases representing common prenatal numerical aberrations (trisomy 21, 18, and 13 and monosomy X) were also successfully diagnosed by CGH. We conclude that CGH is a powerful adjunct to traditional cytogenetic techniques that makes it possible to solve clinical cases of intricate unbalanced aberrations in a single hybridization. CGH may also be a useful adjunct to screen for euchromatic involvement in marker chromosomes. Further technical development may render CGH applicable for routine aberration screening.     

Screening for DMD/BMD deletion carriers by fluorescence in situ hybridization

Fluorescence in situ hybridization (FISH) serves as an excellent alternative for direct detection of heterozygous deletions. Using a set of exon-specific cosmid DNA probes representing 18 exons, one-color FISH on metaphase and interphase preparations was performed to identify Duchenne/Becker muscular dystrophy (DMD/BMD) deletion carriers. The peripheral blood samples from 9 normal male or female controls and 5 females of independent DMD/BMD families, as well as 2 amniotic fluid specimens and 2 chorionic villus samples (CVS) from normal pregnant females, were analyzed. Expected signals were displayed in 72-100% of peripheral blood lymphocyte metaphases or interphases, 60-70% of amniocyte interphases, and 95-99% of chorionic villus cell interphases. One suspected female was identified as a deletion carrier and two were excluded. The results indicated that metaphase and interphase FISH were both useful for detection of heterozygous deletions. FISH, in combination with other available techniques, allowed efficient screening of DMD/BMD deletion carriers. The study also offered preliminary results in support of an approach to prenatal diagnosis of potential fetal carriers.     

Pigmentary mosaicism in hypomelanosis of Ito

We report on a female with mental and motor retardation, facial dysmorphism, abnormal pigmentation reminiscent to hypomelanosis of Ito (HI), and karyotypic mosaicism involving a small supernumerary marker chromosome. The marker chromosome was defined by fluorescence in situ hybridisation (FISH) as a ring X chromosome with breakpoints in the juxtacentromeric region. FISH analysis showed that the ring does not include the XIST locus at the X-inactivation centre and, therefore, may not be subject to X inactivation. X-inactivation studies with the HUMARA (human androgen receptor) and FMR1 assay showed a skewed X-inactivation pattern (85:15) with preferential inactivation of the paternal X chromosome. These results are discussed with respect to the role of functional disomy of Xp in the pathogenesis of HI. Received: 16 February 1998 / Accepted: 17 July 1998     

Study of fluorescence in situ hybridization for detection of chromosome aberration

The authors applied fluorescence in situ hybridization (FISH) technique for the detection of chromosome aberration in interphase nuclei using the probe specific to alphoid repeats on chromosome 11 and X. Chromosome 11 specific probe showed two major spots in lymphocyte nuclei, while X specific probe showed single spot in male and double spots in female respectively. On the other hand three spots were detected in most of the nuclei from HeLa cells with 11 and X specific probes. We concluded that FISH with the use of chromosome specific probe may become a useful and reliable tool for the detection of chromosome aberration in interphase nuclei.     

Del Xq23 in a mosaic Turner female: molecular and cytogenetic studies.

We report a Turner patient aged 22 years with a 45,X/46,X,del(X)(q23) karyotype. Late replication studies showed preferential inactivation of the deleted X chromosome; FISH studies with a probe for total human telomeres showed hybridisation signal in the telomeres on both the normal and the deleted X chromosomes. Microsatellite analysis in the proposita and her family permitted us to conclude to the maternal origin of the deleted X chromosome, and to detect using the marker DXS1106 (Xq22) a probable meiotic recombination event above the breakage point suggesting that the deletion occurred underneath this point.The mild Turner stigmata may be explained by the 45,X cell line, and the gonadal dysgenesis probably by a partial deletion of the gonadal dysgenesis region Xq13-q23 (excluding Xq22).     

Aneuploid epididymal sperm detected in chromosomally normal and Robertsonian translocation-bearing mice using a new three-chromosome FISH method

We present a new method to detect epididymal sperm aneuploidy (ESA) in mice using simultaneous fluorescence in situ hybridization (FISH) with DNA probes specific for mouse chromosomes X, Y and 8. The method was applied to Robertsonian (Rb) translocation (8.14) heterozygotes and homozygotes as well as the chromosomally normal B6C3F1. The sex ratios of sperm did not differ from the expected 1∶1 and the hybridization efficiencies were ≈99.7% for over 60 000 sperm analyzed. Mice heterozygous for Rb (8.14) produced about tenfold higher rates of sperm with chromosome 8 hyperhaploidy than did Rb (8.14) homozygotes or chromosomally normal mice, while frequencies of sperm with hyperhaploidies for chromosomes X and Y were unaffected in all three lines of mice. Hyperhaploid frequencies obtained with the ESA method were consistent with those of the previous testicular FISH method and were validated by published data obtained by conventional cytogenetic analyses (meiotic metaphase II and first cleavage). Thus, the mouse three-chromosome ESA assay together with the previously developed aneuploidy assay for human sperm constitute a promising pair of interspecific biomarkers for comparative studies of the genetic and physiologic mechanisms of the induction and persistence of aneuploidy in male germ cells. Edited by: T. Hassold     

SKY and FISH analysis of radiation-induced chromosome aberrations: a comparison of whole and partial genome analysis

For a retrospective dose estimation of human exposure to ionising radiation, a partial genome analysis is routinely used to quantify radiation-induced chromosome aberrations. For this purpose, fluorescence in situ hybridisation (FISH) with whole chromosome painting probes for selected chromosomes is usually applied covering about 20% of the whole genome. Since genome-wide screening techniques like spectral karyotyping (SKY) and multiplex FISH (mFISH) have been developed the detection of radiation-induced aberrations within the whole genome has now become feasible. To determine the correspondence between partial and whole genome analysis of radiation-induced chromosome aberrations, they were measured comprehensively in this study using in vitro irradiated blood samples from three donors. We were able to demonstrate that comparable results can be detected with both approaches. However, complex aberrations might be misinterpreted by partial genome analysis. We therefore conclude that whole genome analysis by SKY is useful especially in the high dose range to correct aberration data for complex exchange aberrations.     

Method for the molecular cytogenetic visualization of fragile site FRAXA

Fragile X syndrome is one of the most common reasons for human hereditary mental retardation. It is associated with the expansion of CGG repeats in the 5'-untranslated region of the FMR1 gene, which results in the suppression of its expression and the development of the disease. At present, methods based on PCR and Southern blot analysis are used for diagnostics of the fragile X syndrome. The presence of a fragile site FRAXA on the X chromosome is typical for patients with this pathology. We developed a method of visualizing this site in cell cultures obtained from patients using the fluorescent in situ hybridization (FISH) and the combination of two probes. The method allows one to detect five types of signals on the X chromosome, three of which are normal, while two are associated with the emergence of fragile site FRAXA. An analysis of the distribution of all signal types in cell lines from healthy individuals and patients with fragile X syndrome demonstrated that the method allows one to determine differences between lines with a high statistical significance and that it is applicable to detecting cells that are carriers of the syndrome.