The utilization of interphase cytogenetic analysis for the detection of mosaicism

This study describes a method for defining mosaic aneuploidy by interphase cytogenetics based on statistical limits established from control specimens. Fluorescence in situ hybridization (FISH) has been used to detect the number of copies of specific chromosomes in interphase nuclei from placental tissues of diploid controls and mosaic placentas. FISH was performed using probes D7Z1/D7Z2, D9Z1, D10Z1, and D18Z1, all purchased from Oncor, Inc. Statistical analysis of data obtained from diploid controls was used to determine the one-sided upper reference limit and corresponding 95% confidence interval for the proportion of cells with one and three signals for each of the probes used. The one-sided upper reference limits established the lower levels of monosomy and trisomy detectable using each of the four probes. These statistical parameters were then used to interpret the results obtained by FISH applied to the study of term placentas for the confirmation of prenatally diagnosed chromosomal mosaicism.     

An approach for quantitative assessment of fluorescence in situ hybridization (FISH) signals for applied human molecular cytogenetics.

A number of applied molecular cytogenetic studies require the quantitative assessment of fluorescence in situ hybridization (FISH) signals (for example, interphase FISH analysis of aneuploidy by chromosome enumeration DNA probes; analysis of somatic pairing of homologous chromosomes in interphase nuclei; identification of chromosomal heteromorphism after FISH with satellite DNA probes for differentiation of parental origin of homologous chromosome, etc.). We have performed a pilot study to develop a simple technique for quantitative assessment of FISH signals by means of the digital capturing of microscopic images and the intensity measuring of hybridization signals using Scion Image software, commonly used for quantification of electrophoresis gels. We have tested this approach by quantitative analysis of FISH signals after application of chromosome-specific DNA probes for aneuploidy scoring in interphase nuclei in cells of different human tissues. This approach allowed us to exclude or confirm a low-level mosaic form of aneuploidy by quantification of FISH signals (for example, discrimination of pseudo-monosomy and artifact signals due to over-position of hybridization signals). Quantification of FISH signals was also used for analysis of somatic pairing of homologous chromosomes in nuclei of postmortem brain tissues after FISH with "classical" satellite DNA probes for chromosomes 1, 9, and 16. This approach has shown a relatively high efficiency for the quantitative registration of chromosomal heteromorphism due to variations of centromeric alphoid DNA in homologous parental chromosomes. We propose this approach to be efficient and to be considered as a useful tool in addition to visual FISH signal analysis for applied molecular cytogenetic studies.     

Molecular cytogenetic characterization of 17 rob(13q14q) Robertsonian translocations by FISH, narrowing the region containing the breakpoints.

We have characterized 17 rob(13q14q) Robertsonian translocations, using six molecular probes that hybridize to the repetitive sequences of the centromeric and shortarm regions of the five acrocentric chromosomes by FISH. The rearrangements include six de novo rearrangements and the chromosomally normal parents, five maternally and three paternally inherited translocations, and three translocations of unknown origin. The D21Z1/D13Z1 and D14Z1/D22Z1 centromeric alpha-satellite DNA probes showed all rob(13q14q) chromosomes to be dicentric. The rDNA probes did not show hybridization on any of the 17 cases studied. The pTRS-47 satellite III DNA probe specific for chromosomes 14 and 22 was retained around the breakpoints in all cases. However, the pTRS-63 satellite III DNA probe specific for chromosome 14 did not show any signals on the translocation chromosomes examined. In 16 of 17 translocations studied, strong hybridization signals on the translocations were detected with the pTRI-6 satellite I DNA probe specific for chromosome 13. All parents of the six de novo rob(13q14q), including one whose pTRI-6 sequence was lost, showed strong positive hybridization signals on each pair of chromosomes 14 and 13, with pTRS-47, pTRS-63, and pTRI-6. Therefore, the translocation breakpoints in the majority of rob(13q14q) are between the pTRS-47 and pTRS-63 sequences in the p11 region of chromosome 14 and between the pTRI-6 and rDNA sequences within the p11 region of chromosome 13.     

Atomic force microscopy and scanning near-field optical microscopy studies on the characterization of human metaphase chromosomes

A better knowledge of biochemical and structural properties of human chromosomes is important for cytogenetic investigations and diagnostics. Fluorescence in situ hybridization (FISH) is a commonly used technique for the visualization of chromosomal details. Localizing specific gene probes by FISH combined with conventional fluorescence microscopy has reached its limit. Also, microdissecting DNA from G-banded human metaphase chromosomes by either a glass tip or by laser capture needs further improvement. By both atomic force microscopy (AFM) and scanning near-field optical microscopy (SNOM), local information from G-bands and chromosomal probes can be obtained. The final resolution allows a more precise localization compared to standard techniques, and the extraction of very small amounts of chromosomal DNA by the scanning probe is possible. Besides new strategies towards a better G-band and fluorescent probe detection, this study is focused on the combination of biochemical and nanomanipulation techniques which enable both nanodissection and nanoextraction of chromosomal DNA.     

Fluorescence intensity profiles of in situ hybridization signals depict genome architecture within human interphase nuclei

An approach towards construction of two-dimensional (2D) and three-dimensional (3D) profiles of interphase chromatin architecture by quantification of fluorescence in situ hybridization (FISH) signal intensity is proposed. The technique was applied for analysis of signal intensity and distribution within interphase nuclei of somatic cells in different human tissues. Whole genomic DNA, fraction of repeated DNA sequences (Cot 1) and cloned satellite DNA were used as probes for FISH. The 2D and 3D fluorescence intensity profiles were able to depict FISH signal associations and somatic chromosome pairing. Furthermore, it allowed the detection of replicating signal patterns, the assessment of hybridization efficiency, and comparative analysis of DNA content variation of specific heterochromatic chromosomal regions. The 3D fluorescence intensity profiles allowed the analysis of intensity gradient within the signal volume. An approach was found applicable for determination of assembly of different types of DNA sequences, including classical satellite and alphoid DNA, gene-rich (G-negative bands) and gene-poor (G-positive bands) chromosomal regions as well as for assessment of chromatin architecture and targeted DNA sequence distribution within interphase nuclei. We conclude the approach to be a powerful additional tool for analysis of interphase genome architecture and chromosome behavior in the nucleus of human somatic cells. The text was submitted by the authors in English.     

A modified nick translation method used with FISH that produces reliable results with archival tissue sections

Nick translation is used to label DNA and RNA to produce probes for in situ hybridization and Northern and Southern blotting. Fluorescence in situ hybridization (FISH) is a widely applied technique used to determine chromosomal and genetic anomalies in many biological samples. Initially the technique was applied to metaphase preparations, but the usefulness of detecting genetic anomalies in solid tumors in situ has resulted in the development of modified protocols. Formalin fixed paraffin processed tissue sections present novel challenges when applying FISH; the probes must be small (between 200 and 600 base pairs) and pretreatment is necessary before the probes can be applied to tissue sections, to promote probe access to target DNA. Here we report on a modification of a nick translation method to produce a probe that can reliably be used with FISH in paraffin processed tissue sections.     

Severe psychomotor retardation in a boy with a small supernumerary marker chromosome 19p

We describe the clinical case of a nine-year-old boy with psychomotor retardation and a small supernumerary marker chromosome (sSMC) present in mosaic form. Fluorescence in situ hybridization (FISH) using centromere cross-hybridizing probes D1/5/19Z (pZ5.1), the whole chromosome paint probe 19, pool YACs19p (839B1, 872G3, 728C8), and pool YACs19q (767C4, 761C1, 786G6) demonstrated that the sSMC was derived from chromosome 19p. Based on GTG-banding and FISH analyses, the patient's karyotype was interpreted as: 47,XY,+mar.ish der(19) (:p13.3-->p11:)(839B1+, 872G3+,728C8+, D1/5/19Z+) de novo[52]/46,XY[48]. To our knowledge, only two other similar cases have been reported. This case helps to better delineate karyotype-phenotype correlations between sSMC 19p and associated clinical phenomena.     

Simultaneous, multilocus FISH analysis for detection of microdeletions in the diagnostic evaluation of developmental delay and mental retardation.

Many microdeletion and contiguous gene-deletion syndromes include mental retardation as a clinical feature. We have developed MultiFISH, a FISH assay using several probes to simultaneously screen for multiple microdeletion syndromes in patients who present with unexplained devleopmental delay and/or mental retardation. This screening tool can be used to determine whether a particular microdeletion syndrome is involved in the etiology of these clinical phenotypes. In this pilot study we combined probes for the commonly deleted regions of Prader-Willi, Angelman, Williams, Smith-Magenis, and DiGeorge/velocardiofacial syndromes in a single hybridization. The probes were differentially labeled, allowing multicolor detection, and 200 individual samples were screened in a blinded fashion. For all patients found by MultiFISH to have deletions, the deletions were originally identified and/or later confirmed by use of single-probe FISH analysis in our diagnostic cytogenetics laboratory. One patient, who was referred for developmental delay and was shown to have a normal G-banded karyotype, was identified by MultiFISH as having a micro-deletion at the DiGeorge/velocardiofacial commonly deleted region. Forty-six of the 200 total samples were tested for microdeletions by use of single FISH probes in the diagnostic laboratory. Ten of these cases were found to have deletions, and all deletions were subsequently detected by use of MultiFISH screen performed in a blinded fashion. Additionally, for all 200 patients tested by use of MultiFISH, no false-positive deletion results were observed. We demonstrate the ability of this technique to scan for and to identify microdeletions in a proportion of patients whose routine karyotype appears normal yet who are mentally retarded and/or developmentally delayed.     

Unbalanced 1q whole-arm translocation resulting in der(14)t(1;14)(q11-12;p11) in myelodysplastic syndrome

Unbalanced whole-arm translocations (WATs) of the long arm of chromosome 1, resulting in complete trisomy 1q, are chromosomal abnormalities detectable in both solid tumors and hematologic neoplasms. Among the WATs of 1q to acrocentric chromosomes, a few patients with der(1;15) described as a dicentric chromosome have been reported so far, whereas cases of der(1;14) are much rarer. We report on a case of der(1;14) detected as single anomaly in a patient with myelodysplastic syndrome. The aim of our work was to investigate the breakpoints of the (1;14) translocation leading to the der(1;14). Fluorescence in situ hybridization (FISH) experiments have been performed on chromosome preparations from bone marrow aspirate, using specific centromeric probes of both chromosomes, as well as a probe mapping to 1q11 band. FISH results showed that in our patient the derivative chromosome was monocentric with a unique centromere derived from chromosome 14. The breakpoints of the translocation were located in the short arm of chromosome 14 and in the long arm of chromosome 1, between the alphoid D1Z5 and the satellite II domains. The 1q breakpoint was within the pericentromeric region of chromosome 1, which is notoriously an unstable chromosomal region, involved in different chromosomal rearrangements.     

Characterization of 10 marker chromosomes in a prostatic cancer cell line by in situ hybridization.

Marker chromosomes contain potentially valuable information about breakpoints in cancer. However, routine banding procedures, by themselves, provide only limited information about the identity of marker chromosomes. In this study, the use of fluorescence in situ hybridization (FISH) with chromosome-specific centromeric probes and whole-chromosome-specific DNA libraries greatly enhanced the identification of 10 marker chromosomes in the primary prostatic cancer cell line PPC-1. Centromeric probes for chromosomes 1, 2, 3, 4, 10, 12, and 17 and whole-chromosome paint libraries for chromosomes 1, 2, 3, 4, 8, and 12, in conjunction with analysis of G-banded metaphases, allowed the major portion(s) of these 10 PPC-1 marker chromosomes to be defined. The results increase the number of identifiable chromosomal breakpoints in this cell line from 9 to 28 sites.     

Interphase cytogenetics in pathology: principles, methods, and applications of fluorescence in situ hybridization (FISH)

 Characteristic chromosome aberrations have been identified in various tumors. Fluorescence in situ hybridization (FISH) using specific probes that are generated by vector cloning or in vitro amplification and labeled with fluorescent dyes allow for the detection of these genetic changes in interphase cells. This technique, that is also referred to as ”interphase cytogenetics”, can be performed in cytological preparations as well as in sections of routinely formaldehyde-fixed and paraffin-embedded tissue. In cancer research and diagnostics, interphase cytogenetics by FISH is used to detect numerical chromosome changes and structural aberrations, e.g., translocations, deletions, or amplifications. In this technical overview, we explain the principles of the FISH method and provide protocols for FISH in cytological preparations and paraffin sections. Moreover, possible applications of FISH are discussed. Accepted: 22 July 1997     

Molecular cloning of zebra finch W chromosome repetitive sequences: evolution of the avian W chromosome

The zebra finch (Taeniopygia guttata) has a large Z chromosome and highly condensed W chromosome. We used the random amplified polymorphic DNA (RAPD) polymerase chain reaction (PCR) technique to isolate female-specific sequences ZBM1 and ZBM2. Southern blot hybridization to male and female zebra finch genomic DNA suggested that these sequences were located on the W chromosome, although homologous sequences appeared to be autosomal or Z-linked. Fluorescent in situ hybridization (FISH) using bacterial artificial chromosome (BAC) clones corresponding to ZBM sequences showed hybridization to the whole W chromosome, suggesting that the BACs encode sequences that are repeated across the entire W chromosome. Based on the sequencing of a ZBM repetitive sequence and Z chromosome derived BAC clones, we demonstrate a random distribution of repeat sequences that are specific to the W chromosome or encoded by both Z and W. The positions of ZW-common repeat sequences mapped to a noncoding region of a Z chromosome BAC clone containing the CHD1Z gene. The apparent lineage-specificity of W chromosome repeat sequences in passerines and galliform birds suggest that the W chromosome had not differentiated well from the Z at the time of divergence of these lineages. Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

An improved method for generating BAC DNA suitable for FISH

Fluorescence in situ hybridization (FISH) is commonly used to identify chromosomal aberrations such as translocations, deletions, duplications, gene fusions, and aneuploidies. It relies on the hybridization of fluorescently labeled DNA probes onto denatured metaphase chromosomes or interphase nuclei. These probes are often generated from DNA sequences cloned within bacterial artificial chromosomes (BACs). Growing these BACs in adequate amounts for FISH can be demanding. We describe FISH performed with bacteriophage Phi29 DNA polymerase amplified BAC DNA. Generating this material required significantly smaller cultures and less time than standard methods. The FISH results obtained were comparable with those obtained from standard BAC DNA. We believe this method of BAC DNA generation is useful for the entire FISH community as it improves considerably on prior methods.     

Assessing chromosomal abnormalities in two-cell bovine in vitro-fertilized embryos by using fluorescent in situ hybridization with three different cloned probes

The aim of this study was to assess the efficiency of fluorescent in situ hybridization (FISH) for detecting chromosomal abnormalities in in vitro-fertilized (IVF) bovine embryos as early as the 2-cell stage. Three different cloned probes were used, two derived from a unique sequence specific to the subtelomeric (D1S48) or subcentromeric regions (19C10) of chromosome 1 and the third (H1A clone) derived from a repetitive sequence that hybridizes to the subcentromeric regions of three other chromosomes (14, 20, 25). Our results show that the incidence of chromosomal abnormalities in 2-cell bovine IVF embryos varied from 28% to 44% according to the probes used for the analysis. Whereas the efficiency of FISH was high with somatic nuclei, it appeared to be highly variable with the 2-cell embryos. FISH efficiency depended firstly on the probe sequence (repetitive or unique sequence), secondly on the chromosomal target region (centromeric or telomeric regions), and thirdly on the embryo cell cycle phase. With a unique sequence probe (19C10) specific to the subcentromeric regions, FISH efficiency was better on nuclei in the S-phase cycle than on those in the G-phase. In S-phase 2-cell embryos, the overall incidence of chromosomal abnormalities was more accurately assessed. It reached 13% and was represented by 1n/2n mixoploidies.     

LNA probes substantially improve detection of bacterial endosymbionts in whole mount of insects by fluorescent in-situ hybridization

ABSTRACT: BACKGROUND: Detection of unculturable bacteria and their localization in the host, by fluorescent in-situ hybridization (FISH), is a powerful technique in the study of host-bacteria interaction. FISH probes are designed to target the 16 s rRNA region of the bacteria to be detected. LNA probes have recently been used in FISH studies and proven to be more efficient. To date no report has employed LNA probes for FISH detection of bacterial endosymbiont in the whole mount tissues. Further, though speculated, bacteriocytes have not been reported from males of Bemisia tabaci. RESULTS: In this study, we compared the efficiency in detecting bacteria by fluorescent DNA oligonucleotides versus modified probes containing Locked Nucleic Acid (LNA) substitution in their structure. We used the insect Bemisia tabaci as the experimental material since it carried simultaneous infection by two bacteria: one a primary endosymbiont, Portiera (and present in more numbers) while the other a secondary endosymbiont Arsenophonus (and present in less numbers). Thus a variation in the abundance of bacteria was expected. While detecting both the bacteria, we found a significant increase in the signal whenever LNA probes were used. However, the difference was more pronounced in detecting the secondary endosymbiont, wherein DNA probes gave weak signals when compared to LNA probes. Also, signal to noise ratio for LNA probes was higher than DNA probes. We found that LNA considerably improved sensitivity of FISH, as compared to the commonly used DNA oligonucleotide probe. CONCLUSION: By employing LNA probes we could detect endosymbiotic bacteria in males, which have never been reported previously. We were able to detect bacteriocytes containing Portiera and Arsenophonus in the males of B. tabaci. Thus, employing LNA probes at optimized conditions will help to significantly improve detection of bacteria at the lowest concentration and may give a comprehensible depiction about their specific distribution within samples.     

Spectral karyotyping, a 24-colour FISH technique for the identification of chromosomal rearrangements

 Spectral karyotyping (SKY) is a new fluorescence in situ hybridisation (FISH) technique that refers to the molecular cytogenetic analysis of metaphase preparations by means of spectral microscopy. For SKY of human metaphase chromosomes, 24 chromosome-specific painting probes are used in just one FISH experiment. The probes are labelled by degenerate oligonucleotide-primed PCR using three fluorochromes and two haptens. Each probe is differentially labelled with one, two, three or four fluorescent dyes, resulting in a unique spectral signature for every chromosome. After in situ hybridisation and immunodetection, a spectral image is acquired using a conventional fluorescence light microscope equipped with a custom-designed triple-bandpass filter and the SpectraCube, which is able to retrieve spectral information for every pixel in a digital CCD image. The 24-colour display and chromosome classification are based on the unique emission spectra of the chromosomes. Together with chromosome banding information from an inverted DAPI or a G-banded metaphase, a comprehensive overview of chromosomal aberrations is presented. Accepted: 3 July 1997     

Localization of DNA probes for human ribosomal genes on barley chromosomes]

To estimate the possibility of plant genome mapping using human genome probes, the probes fluorescent in situ hybridization (FISH) of human 18S-28S rDNA (clon 22F9 from the LA-13NCO1 library) was carried out on chromosomes of the spring barley Hordeum vulgare L. As a control, wheat rDNA probe (clon pTa71) was taken. Hybridization of the wheat DNA probe revealed two major labelling sites on mitotic barley chromosomes 5I (7H) and 6I (6H), as well as several minor sites. With the human DNA probe, signals were detected in the major sites of the ribosomal genes on chromosomes 5I (7H) and 6I (6H) only when the chromosome preparations were obtained using an optimized technique with obligatory pepsin treatment followed by hybridization. Thus, this study demonstrates that physical mapping of plant chromosomes with human DNA probes that are 60 to 75% homologous to the plant genes is possible. It suggests principal opportunity for the FISH mapping of plant genomes using probes from human genome libraries, obtained in the course of the total sequencing of the human genomes and corresponding to the coding regions of genes with known functions.     

High-temperature fluorescent in situ hybridization for detecting Escherichia coli in seawater samples, using rRNA-targeted oligonucleotide probes and flow cytometry

Fluorescence in situ hybridization (FISH) is a widely used method to detect environmental microorganisms. The standard protocol is typically conducted at a temperature of 46 degrees C and a hybridization time of 2 or 3 h, using the fluorescence signal intensity as the sole parameter to evaluate the performance of FISH. This paper reports our results for optimizing the conditions of FISH using rRNA-targeted oligonucleotide probes and flow cytometry and the application of these protocols to the detection of Escherichia coli in seawater spiked with E.coli culture. We obtained two types of optimized protocols for FISH, which showed rapid results with a hybridization time of less than 30 min, with performance equivalent to or better than the standard protocol in terms of the fluorescence signal intensity and the FISH hybridization efficiency (i.e., the percentage of hybridized cells giving satisfactory fluorescence intensity): (i) one-step FISH (hybridization is conducted at 60 to 75 degrees C for 30 min) and (ii) two-step FISH (pretreatment in a 90 degrees C water bath for 5 min and a hybridizing step at 50 to 55 degrees C for 15 to 20 min). We also found that satisfactory fluorescence signal intensity does not necessarily guarantee satisfactory hybridization efficiency and the tightness of the targeted population when analyzed with a flow cytometer. We subsequently successfully applied the optimized protocols to E. coli-spiked seawater samples, i.e., obtained flow cytometric signatures where the E. coli population was well separated from other particles carrying fluorescence from nonspecific binding to probes or from autofluorescence, and had a good recovery rate of the spiked E. coli cells (90%).     

Fluorescence in situ hybridization (FISH) on human chromosomes using photoprobe biotin-labeled probes.

Fluorescence in situ hybridization (FISH) on human chromosomes in meta- and interphase is a well-established technique in clinical and tumor cytogenetics and for studies of evolution and interphase architecture. Many different protocols for labeling the DNA probes used for FISH have been published. Here we describe for the first time the successful use of Photoprobe biotin-labeled DNA probes in FISH experiments. Yeast artificial chromosome (YAC) and whole chromosome painting (wcp) probes were tested.