Chromosome Painting - an overview (2023)

Fluorescence In Situ Hybridization

In FISH, fluorescently labeled DNA probes are hybridized to interphase nuclei or metaphase spreads prepared for standard cytogenetic analysis. FISH can also be applied to a wide range of cellular preparations such as banded slides, air-dried bone marrow or blood smears, fresh tumor touch prints, frozen or paraffin-embedded tissue sections, or nuclear isolates from fresh or fixed tissues.

A variety of FISH probes, each targeting a specific region or the entire chromosome, are available. Probes routinely used in the analysis of hematologic malignancies include chromosome-specific enumerator (i.e., mostly centromeric) probes, gene- or locus-specific probes, whole chromosome painting probes, arm-specific sequence probes, and telomeric probes.

Chromosome-specific centromeric probes are derived from the highly repetitive mostly alpha-satellite DNA sequences located within the centromeres. Because the target size is several hundred kilobases (kb) in length, the probes exhibit bright, discrete signals and are easy to evaluate in both metaphase and interphase nuclei. Centromeric probes are useful in identifying numerical abnormalities (aneuploidy), dicentric chromosomes, and the origin of marker chromosomes. Clinically important aberrations such as trisomy 12 in CLL (seeFig. 7-2,B), monosomy 7 in AML, and high hyperploidy in ALL—all of which are detected at a lower incidence by conventional cytogenetics owing to low mitotic index or poor morphology—are routinely evaluated by FISH in many clinical laboratories. Another example is the use of differentially labeled probes specific for chromosomes X and Y in monitoring engraftment in sex-mismatched allogeneic stem cell transplantation.

Whole chromosome painting probes (WCP) or arm-specific sequence probes use mixtures of fluorescently labeled DNA sequences derived from the entire length of the specific chromosome or one of its arms.^26,27 They are helpful in characterizing complex rearrangements and marker chromosomes. However, cryptic rearrangements affecting terminal regions may remain undetected, because of suppression of the repetitive DNA sequences within these regions. The application of chromosome painting probes is limited to metaphase analysis because the signals are often large and diffuse in interphase. Chromosome-specific telomeric or subtelomeric probes are derived from DNA sequences located at or adjacent to the telomeres and are effective in detecting terminal, interstitial, and cryptic translocations that are below the resolution of conventional cytogenetics and/or are undetectable by WCP probes.

Gene-specific or locus-specific probes are derived from unique DNA sequences or loci within the chromosome. With banding techniques on highly extended chromosomes, the smallest detectable chromosome abnormality is 2000 to 3000 kb, whereas gene- or locus-specific probes can routinely detect regions as small as 0.1 kb.²⁷ As such, these probes have wide application in both basic and clinical research. Gene-specific or locus-specific probes have been extremely useful in gene mapping and in defining structural rearrangements, amplifications, and origin of marker chromosomes in both metaphase chromosomes and interphase nuclei.

Abstract

Chromosome painting describes a range of techniques that employ fluorescently labeled DNA probes to characterize chromosomal rearrangements. These probes paint the entire length or part of a target chromosome, either in a single color or in a characteristic banding pattern. Furthermore, chromosome paints allow the identification of multiple chromosomes using a panel of fluorescence dyes, or through the use of computer enhancement. When used in conjunction with other approaches, chromosome painting can allow the characterization of unidentified chromosomal regions. These approaches have considerable application in the identification and characterization of abnormal chromosomes in a plethora of species. Herein, the basic principles of chromosome painting are described, along with a variety of related techniques, their application and sources of further reading.

Fluorescence In Situ Hybridization

In situ hybridization (ISH) methods utilize the fact that DNA is organized into two antiparallel complementary strands. Thus, if the two strands are denatured (separated) through heating, a single-stranded probe can bind its complementary target. At fluorescence ISH (FISH), the probes have been labelled directly or indirectly by fluorophores, allowing for detection by fluorescence microscopy.⁸⁷ Sequences ranging in size from approximately 10 kb up to entire chromosomes can be visualized with FISH probes, and by using different fluorophores for different chromosomes, the entire genome can be studied (multicolor FISH, spectral karyotyping). FISH is thus highly versatile; “painting probes” label entire or parts of chromosomes, locus-specific probes target individual genes or unique sequences, and repeat sequence probes label specific chromosomal structures present in multiple copies (e.g., centromeres, telomeres). The probes can also be labeled with nonfluorescent haptens that can be used for secondary detection by enzymatic methods, so-called chromogenic ISH (CISH), of particular use for analysis of fixed tissue sections.⁸⁸ More important clinically, ISH analyses can be performed on both dividing cells (metaphase;Fig. 4.4A and B) and nondividing cells (interphase;Fig. 4.4C and D). Thus, in contrast to chromosome banding, prior culturing is not needed. Furthermore, ISH can be successfully performed on minute samples and thus is well suited for analysis of cells from fine- or core-needle biopsies. In addition, the technique can provide rapid results (within 1 to 2 days).

ISH, especially FISH with locus-specific probes, has thus become a robust and useful ancillary method in soft tissue tumor pathology. It is particularly useful for detecting gene rearrangements by “break-apart probes”; the status of the gene in question is queried by probes that flank the gene, typically with one end labeled in red and the other in green. If the gene locus is intact, the two signals remain close to each other and are perceived as a yellow signal, but if the gene is affected by a structural rearrangement such as a translocation or inversion, the probe is split and seen as separate red and green signals (Fig. 4.4C). To increase specificity and stringency, separate probes for the two partners in a gene fusion could be labeled in red and green, giving rise to yellow fusion signals when positive (Fig. 4.4D). Locus-specific probes can also be used to detect deletions and amplifications (Fig. 4.4A). Reliable FISH probes are now commercially available for many clinically relevant gene fusions and amplicons in soft tissue tumors.

FISH on FFPE tissue sections poses technical problems not encountered when using cell spreads or imprint slides. First, the chemical procedures involved in the fixation of tumor tissue damage the DNA, reducing the stringency of the hybridization. Second, as an effect of cells not being neatly separated invivo and thus possibly overlapping each other, and because some nuclei are cut when the sections are prepared, the cutoff levels for false-positive and false-negative signals could be quite high.

See also:

BAC (Bacterial Artificial Chromosome); Candidate Gene; Chiasma; Chromosome Painting; Comparative Genomic Hybridization (CGH); Contig; CpG Islands; Crossing-Over; FISH (Fluorescent in situ Hybridization); Functional Genomics; Haldane, J.B.S.; Human Genome Project; In situ Hybridization; Linkage Disequilibrium; LOD Score; Mapping Function; Marker; Microarray Technology; Morgan, Thomas Hunt; Physical Mapping; Polytene Chromosomes; QTL Mapping; QTL (Quantitative Trait Locus)Restriction Fragment Length Polymorphism (RFLP); Single Nucleotide Polymorphisms (SNPs); YAC (Yeast Artificial Chromosome)

98.1

Methods of Chromosome Analysis

Cytogenetic studies are usually performed on peripheral blood lymphocytes, although cultured fibroblasts obtained from a skin biopsy may also be used. Prenatal (fetal) chromosome studies are performed with cells obtained from the amniotic fluid (amniocytes), chorionic villus tissue, and fetal blood or, in the case of preimplantation diagnosis, by analysis of ablastomere (cleavage stage) biopsy, polar body biopsy, or blastocyst biopsy. Cytogenetic studies of bone marrow have an important role in tumor surveillance, particularly among patients with leukemia. These are useful to determine induction of remission and success of therapy or in some cases the occurrence of relapses.

Chromosome anomalies include abnormalities of number and structure and are the result of errors during cell division. There are 2 types of cell division: mitosis, which occurs in most somatic cells, and meiosis, which is limited to the germ cells. Inmitosis, 2 genetically identical daughter cells are produced from a single parent cell. DNA duplication has already occurred duringinterphase in the S phase of the cell cycle (DNA synthesis). Therefore, at the beginning of mitosis the chromosomes consist of 2 double DNA strands joined together at the centromere, known assister chromatids. Mitosis can be divided into 4 stages: prophase, metaphase, anaphase, and telophase.Prophase is characterized by condensation of the DNA. Also during prophase, the nuclear membrane and the nucleolus disappear and the mitotic spindle forms. Inmetaphase the chromosomes are maximally compacted and are clearly visible as distinct structures. The chromosomes align at the center of the cell, and spindle fibers connect to the centromere of each chromosome and extend to centrioles at the 2 poles of the mitotic figure. Inanaphase the chromosomes divide along their longitudinal axes to form 2 daughter chromatids, which then migrate to opposite poles of the cell.Telophase is characterized by formation of 2 new nuclear membranes and nucleoli, duplication of the centrioles, and cytoplasmic cleavage to form the 2 daughter cells.

Meiosis begins in the female oocyte during fetal life and is completed years to decades later. In males it begins in a particular spermatogonial cell sometime between adolescence and adult life and is completed in a few days. Meiosis is preceded by DNA replication so that at the outset, each of the 46 chromosomes consists of 2 chromatids. In meiosis, adiploid cell (2n = 46 chromosomes) divides to form4 haploid cells (n = 23 chromosomes). Meiosis consists of 2 major rounds of cell division. Inmeiosis I, each of the homologous chromosomes pair precisely so thatgenetic recombination, involving exchange between 2 DNA strands (crossing over), can occur. This results in reshuffling of the genetic information for the recombined chromosomes and allows further genetic diversity. Each daughter cell then receives 1 of each of the 23 homologous chromosomes. In oogenesis, one of the daughter cells receives most of the cytoplasm and becomes the egg, whereas the other smaller cell becomes the first polar body.Meiosis II is similar to a mitotic division but without a preceding round of DNA duplication (replication). Each of the 23 chromosomes divides longitudinally, and the homologous chromatids migrate to opposite poles of the cell. This produces 4 spermatogonia in males, or an egg cell and a 2nd polar body in females, each with a haploid (n = 23) set of chromosomes. Consequently, meiosis fulfills 2 crucial roles: It reduces the chromosome number from diploid (46) to haploid (23) so that on fertilization a diploid number is restored, and it allows genetic recombination.

2.5 Other Enabling Technologies

Other technologies have improved the detection and interpretation of various toxicity endpoints, including cytogenetics using chromosome painting (via fluorescence in situ hybridisation [FISH], to detect and localise the presence or absence of specific DNA sequences), the use of quantum dot cell imaging (9) and the detection of biomarkers of endogenous exposure and effect. Biomarkers, together with highly sensitive analytical techniques, such as accelerated mass spectrometry (AMS), and the availability of non-invasive ‘real-time’ diagnostic imaging, have contributed to enabling earlier than usual first-in human studies of earlier first-in human studies (10).

Toxicokinetics can provide useful information for interpreting hazard data for risk assessment, by: (a) relating external and internal dose effects; and (b) facilitating interspecies extrapolation. In its more-elaborate form, physiologically based pharmacokinetic (PBPK) modelling involves representing organs or groups of organs as discrete compartments interconnected with physiological volumes and blood flows into and out of which test chemicals and their metabolites partition, before being eliminated (11). Invitro biokinetics applies similar principles to tissue culture systems, to study test material availability to the target site, facilitating invitro to invivo extrapolation (12).

Finally, the vast amount of data generated by these collective approaches, and especially genomics, is being searched, analysed, compared and interpreted by using increasingly powerful bioinformatics procedures. These involve a range of computerised and statistical sorting, and cataloguing techniques.

2.4.2 Random position of chromosome territory

The chromosomes of A. thaliana do not show the Rabl configuration (Lysak et al., 2001; Pecinka et al., 2004). Chromosome painting using chromosome-specific BACs (bacterial artificial chromosomes) as probes has been conducted to get insight into the 3D structure of chromosomes in the nuclei of A. thaliana (Berr and Schubert, 2007; Berr et al., 2006; Lysak et al., 2001; Pecinka et al., 2004). These analyses revealed that the positioning of chromosome territories in interphase nuclei is random except for chromosomes with nucleolus-organizing regions. This random positioning of chromosome territories is different from the preferential arrangement of chromosome territories in mammals (Cremer et al., 2001) and birds (Habermann et al., 2001). In their chromosome territory organization, gene-poor and large chromosomes occupy more peripheral regions than the preferential arrangement of gene-dense or small chromosomes.

Because FISH and GISH experiments require the fixation of cell nuclei and chromosomes and high-temperature treatment for hybridization, they cannot be adapted to live cell imaging. To overcome this problem, the lacO/LacI-GFP system, which allows tracking of specific gene loci, has been developed as a powerful method for detecting the in vivo dynamics of chromatins in plant interphase nuclei (Matzke et al., 2010). The system contains lac repressor (LacI) fused with GFP and tandem repeats of lac operator (lacO) integrated into the chromosome. Although long tandem repeats of lacO alter the arrangement of chromatins in interphase nuclei (Jovtchev et al., 2008, 2011), short repeats of lacO with LacI-GFP allow monitoring of chromatin dynamics in living plant cells (Jovtchev et al., 2011; Matzke et al., 2010). This in vivo tracking technique will help reveal spatiotemporal dynamics under various environmental conditions in plants.

DNA Probes Used in FISH

Total genomic probes are prepared by labeling DNA extracted from blood samples, cell cultures, or solid tissues. Chromosomes hybridized with these probes show an evenly distributed signal along their length, referred to as ‘chromosome painting’. The main application of total genomic probes has been in the identification of human chromosome material in human-to-rodent interspecific somatic cell hybrids, including radiation-reduced cell hybrids.

‘Chromosome-specific paint probes’ are genomic probes that were prepared initially from chromosome-specific genomic libraries cloned in plasmid vectors. They can also be made from single-chromosome interspecific somatic cell hybrids. Most are now prepared from flow-sorted chromosomes and these tend to have the highest resolution. Each chromosome-specific paint is made from sorting 300–500 chromosomes with a dual-laser flow cytometer, and amplifying chromosomal DNA fragments by the random-primed polymerase chain reaction (DOP-PCR). Flow-sorted chromosomes can be obtained in high purity, and the PCR procedure amplifies over 90% of the chromosomal DNA. Chromosome-specific hybridization, free of background signal, is assured by prehybridization of the probe with itself before application to the test material. This ensures that highly repetitive signals are largely eliminated, and unique, conserved DNA sequences are available to paint all but the heterochromatic regions of the chromosomes. Chromosome-specific paint probes have wide application in the analysis of complex chromosomal aberrations and are commercially available from several distributors either as single chromosome-specific paint probes or as complete probe sets in which each chromosome is labeled differently for multiplex FISH (M-FISH) analysis. This allows the analysis of a complete cell in one hybridization step.

The main disadvantage of chromosome-specific paint probes is that they are unable to identify intrachromosomal aberrations such as inversions, duplications, and insertions, and that areas containing repetitive sequences, especially telomeres and centromeres, are not painted. In these cases, region-specific paint probes prepared from amplified chromosome segments obtained by chromosome microdissection have found some application.

Chromosome-specific centromeric probes are prepared from cloned alphoid repeat sequences, which are located adjacent to centromeres. Almost all human chromosomes have chromosome-specific sequences of this type. The exceptions are chromosomes 13, 14, 21, and 22. Chromosomes 13 and 21 have the same centromeric sequences, different from 14 and 22, which also share the same sequences. These probes are used to determine chromosome copy number in interphase nuclei. More than 80% of normal diploid nuclei will show two distinct signals when hybridized with a chromosome-specific centromeric probe. Centromeric probes are therefore used for aneuploidy detection in uncultured amniotic fluid cells, for preimplantation diagnosis in cells from the blastocyst, for the detection of residual disease in the management of certain hematological malignancies, and for the analysis of nondisjunctional abnormalities in sperm. Chromosome-specific sequences cloned in yeast artificial chromosome (YAC), bacterial artificial chromosome (BAC), or cosmid vectors replace the lack of specific centromeric probes for aneuploidy detection involving chromosomes 13, 14, 21, and 22.

The project to map and sequence the human genome had, as one of its by-products, a complete series of overlapping DNA clones from which reference probes can be produced, which can be used as FISH markers to delineate any point on any chromosome. Cloned in BACs and other vectors, they can be used to characterize specific break points and to detect specific microdeletions (such as the DiGeorge syndrome on chromosome 22). These single-copy DNA sequence probes have wide application in clinical cytogenetics and in the mapping and cloning of disease genes.

Telomere-specific probes are available for the ends of all human chromosomes. They have proved to be particularly valuable in the detection of reciprocal translocations which are beyond the resolution of conventional diagnostic cytogenetics.

3.2 FISH

FISH can be used to identify chromosomal abnormalities via labeled probes that target-specific DNA sequences. More than one probe may be used at the same time, that is, each probe labeled with a different fluorochrome. The most useful FISH probes are: centromeric, chromosome painting, and locus-specific for fusion, deletion or duplication studies.

Centromeric enumeration probes hybridize to the alpha (or beta) satellite repeat sequences within the centromeric region specific to each chromosome. As such, these probes are used for chromosome enumeration, that is, detection of ploidy abnormalities. Chromosome painting probes, generated from chromosome-specific probe libraries, are useful in deciphering cytogenetic aberrations. Locus-specific probes hybridize to a unique human genome sequences. They are most frequently used to detect rearrangements, gains, and deletions as well as amplification in both metaphase and interphase cells. FISH interphase analysis is an attractive and practical way to assess ERBB2 amplification [192].

Evaluation of the tumor and its CTC by FISH has revealed that a negative ERBB2 primary lesion can release positive ERBB2 CTC and vice-versa [149].

Chromosomal Translocation

Specific chromosomal translocations can serve as useful diagnostic markers for some types of neoplasms. For instance, translocation of a portion of the bcl-2 proto-oncogene to the immunoglobulin heavy chain gene locus is common in follicular non-Hodgkin's lymphoma (NHL) and is a clinically useful genetic marker for this neoplasm. This translocation results in the overexpression of BCL-2 protein to inhibit apoptosis in tumor cells.

Chromosome translocations can also result in the actual fusion of two different genes to produce a novel chimeric protein (fusion protein) that is not normally present in human cells. Chimeric proteins often have new and abnormal functions (Fig. 5-15). Many fusion proteins act as transcription factors that can activate novel promoters throughout the genome to cause extensive and complex alterations in gene expression. Since these hybrid proteins can activate (or repress) the expression of many different genes, a single translocation event can cause global changes in gene expression that can directly transform a normal cell to produce a malignancy.

Fusion proteins resulting from translocation occur most frequently in hematopoietic neoplasms, a subset of pediatric neoplasms, and sarcomas. One of the first neoplastic translocations identified is a marker chromosome (the so-called Philadelphia chromosome) in patients with chronic leukemia. Tumors that express chimeric proteins are often defined diagnostically by the presence of these specific translocation events. For example, the pathologic diagnosis of acute promyelocytic leukemia (a variant of myeloid leukemia) now rests on the demonstration of a translocation between the retinoic acid receptor (RAR) protein gene and the PML gene. Treatment of patients who have leukemias expressing this fusion protein with all-trans-retinoic acid can induce clinical remission in a high percentage. Malignant neoplasms resulting from single translocation events tend to retain intact apoptotic pathways and are often more responsive to cancer treatment than are many carcinomas. Since most carcinomas develop by the sequential accumulation of numerous genetic alterations, they often have defective apoptotic pathways and are resistant to many forms of therapy.