Spectral karyotyping refines cytogenetic diagnostics of constitutional chromosomal abnormalities

Karyotype analysis by chromosome banding is the standard method for identifying numerical and structural chromosomal aberrations in pre- and postnatal cytogenetics laboratories. However, the chromosomal origins of markers, subtle translocations, or complex chromosomal rearrangements are often difficult to identify with certainty. We have developed a novel karyotyping technique, termed spectral karyotyping (SKY), which is based on the simultaneous hybridization of 24 chromosome-specific painting probes labeled with different fluorochromes or fluorochrome combinations. The measurement of defined emission spectra by means of interferometer-based spectral imaging allows for the definitive discernment of all human chromosomes in different colors. Here, we report the comprehensive karyotype analysis of 16 samples from different cytogenetic laboratories by merging conventional cytogenetic methodology and spectral karyotyping. This approach could become a powerful tool for the cytogeneticists, because it results in a considerable improvement of karyotype analysis by identifying chromosomal aberrations not previously detected by G-banding alone. Advantages, limitations, and future directions of spectral karyotyping are discussed. Received: 4 August 1997 / Accepted: 8 September 1997     

Characterization of chromosomal aberrations in diffuse large B-cell lymphoma (DLBL) by G-banding and spectral karyotyping (SKY)

Cytogenetic chromosome analysis by classical G-banding was supplemented by spectral karyotyping (SKY) in 12 cases of diffuse large B-cell lymphoma (DLBL). SKY is a fluorescence in-situ-based, genome-wide screening technique allowing identification of genetic material even in highly condensed metaphase chromosomes of poor morphology. By simultaneous hybridization of whole chromosome painting probes onto tumor chromosome spreads genetic rearrangements are visualized permitting the clarification of even complex karyotype alterations and the identification of genetic material of previously unknown origin, so-called marker chromosomes. Taking the SKY results into account, we reevaluated the G-banding karyotypes initially carried out, thus generating a more precise karyotype in ten of twelve (83%) cases investigated. In particular, thirteen chromosomal rearrangements not correctly recognized by classical cytogenetics were identified, the genetic origin of seven marker chromosomes was elucidated and three structural genetic rearrangements were redefined. We found SKY to be a valuable technique to establish a definite karyotype in addition to classical cytogenetics.     

Spectral Karyotyping to Study Chromosome Abnormalities in Humans and Mice with Polycystic Kidney Disease

Conventional method to identify and classify individual chromosomes depends on the unique banding pattern of each chromosome in a specific species being analyzed ^{1, 2}. This classical banding technique, however, is not reliable in identifying complex chromosomal aberrations such as those associated with cancer. To overcome the limitations of the banding technique, Spectral Karyotyping (SKY) is introduced to provide much reliable information on chromosome abnormalities.SKY is a multicolor fluorescence in-situ hybridization (FISH) technique to detect metaphase chromosomes with spectral microscope ^{3, 4}. SKY has been proven to be a valuable tool for the cytogenetic analysis of a broad range of chromosome abnormalities associated with a large number of genetic diseases and malignancies ^{5, 6}. SKY involves the use of multicolor fluorescently-labelled DNA probes prepared from the degenerate oligonucleotide primers by PCR. Thus, every chromosome has a unique spectral color after in-situ hybridization with probes, which are differentially labelled with a mixture of fluorescent dyes (Rhodamine, Texas Red, Cy5, FITC and Cy5.5). The probes used for SKY consist of up to 55 chromosome specific probes ^7-10.The procedure for SKY involves several steps (Figure 1). SKY requires the availability of cells with high mitotic index from normal or diseased tissue or blood. The chromosomes of a single cell from either a freshly isolated primary cell or a cell line are spread on a glass slide. This chromosome spread is labeled with a different combination of fluorescent dyes specific for each chromosome. For probe detection and image acquisition,the spectral imaging system consists of sagnac interferometer and a CCD camera. This allows measurement of the visible light spectrum emitted from the sample and to acquire a spectral image from individual chromosomes. HiSKY, the software used to analyze the results of the captured images, provides an easy identification of chromosome anomalies. The end result is a metaphase and a karyotype classification image, in which each pair of chromosomes has a distinct color (Figure 2). This allows easy identification of chromosome identities and translocations. For more details, please visit Applied Spectral Imaging website (http://www.spectral-imaging.com/).SKY was recently used for an identification of chromosome segregation defects and chromosome abnormalities in humans and mice with Autosomal Dominant Polycystic Kidney Disease (ADPKD), a genetic disease characterized by dysfunction in primary cilia ^11-13. Using this technique, we demonstrated the presence of abnormal chromosome segregation and chromosomal defects in ADPKD patients and mouse models ¹⁴. Further analyses using SKY not only allowed us to identify chromosomal number and identity, but also to accurately detect very complex chromosomal aberrations such as chromosome deletions and translocations (Figure 2).     

Stylized chromosome images

Stylized chromosome images 1) serve as a format to test effects of preprocessing algorithms used in automated karyotyping; 2) enhance the ability of humans to perform quantitative analysis of chromosomal aberrations; 3) provide an alternative format for karyotype hard copies produced by automated systems. Stylized chromosomes are two-dimensional computer-generated images based on information extracted from one-dimensional width and density profiles. These profiles correspond to what cytogeneticists observe through the microscope as the shape and banding patterns of stained chromosomes. Stylized presentation sharpens chromosome band boundaries and perimeters, reduces "noise," and enhances gray level variations, which are difficult to distinguish by humans on photographic or computer generated karyotypes. Karyotyping accuracy using stylized images was used to detect difficult areas for automated chromosome identification. Landmark bands sufficient to classify chromosomes were identified; shapes of chromosomes reflected in width profiles were said to aid classification. A two-step automated karyotyping strategy proposed is: 1) classify chromosomes by landmarks, minimum information needed for identification; 2) subsequently employ the full banding pattern with maximum resolution to detect aberrations. Stylized images of abnormal chromosomes have potential for testing hypothesis regarding breakpoints and quantitative analysis, but improvements are needed in homologue normalization and definition of termini of chromosomes.     

The interrelationships of chromosome banding patterns in procyonids, viverrids, and felids.

The Giemsa banding patterns of the chromosomes of 4 species of Procyonidae, 13 of Viverridae, and 6 of Felidae have been studied. The patterns have all been related to the basic felid karyotype. Each procyonid and viverrid has a distinctive karyotype, whereas some felids have indistinguishable karyotypes even when banded. Extensive interfamilial G-banding conservatism has been demonstrated, with many chromosomes being "shared" by all three families. A chromosome identification and karyotyping system for the Carnivora has been devised to facilitate comparison of data from future studies. Phylogenetic implications of the findings have been discussed.     

The Bombyx mori karyotype and the assignment of linkage groups

Lepidopteran species have a relatively high number of small holocentric chromosomes (Bombyx mori, 2n = 56). Chromosome identification has long been hampered in this group by the high number and by the absence of suitable markers like centromere position and chromosome bands. In this study, we carried out fluorescence in situ hybridization (FISH) on meiotic chromosome complements using genetically mapped B. mori bacterial artificial chromosomes (BACs) as probes. The combination of two to four either green or red fluorescence-labeled probes per chromosome allowed us to recognize unequivocally each of the 28 bivalents of the B. mori karyotype by its labeling pattern. Each chromosome was assigned one of the already established genetic linkage groups and the correct orientation in the chromosome was defined. This facilitates physical mapping of any other sequence and bears relevance for the ongoing B. mori genome projects. Two-color BAC-FISH karyotyping overcomes the problem of chromosome recognition in organisms where conventional banding techniques are not available.     

A complex chromosome rearrangement involving chromosome 8, 11, and 12 analyzed by conventional cytogenetic investigations, fluorescence in situ hybridisation, and spectral karyotyping.

We report on a 29-year-old woman with a history of five spontaneous abortions and a balanced complex chromosome rearrangement (CCR) involving break points between chromosomes 8, 11, and 12. Fluorescence in situ hybridisation (FISH) in combination with giemsa trypsin banding techniques were essential for the identification of the breakpoints. In addition, the results were confirmed by 24-colour FISH using the spectral karyotyping system (SKY). The karyotype was 46,XX,t(8;11;12)(8qter-->8p10::12p10-->12pter;11pter--> 11q14::8p10-->8pter;12qter-->12p10::11q14-->11qter). Application of SKY facilitated detection of all three chromosomes involved and supported the localisation of the breakpoints by a single time and sample saving investigation.     

Multicolor-FISH applied to resolve complex chromosomal changes in a case of T-ALL (FAB L2)

We report on a patient with a clinically diagnosed acute lymphoblastic leukemia (ALL) with partial unrecorded complex translocation events especially involving chromosomes 5, 9 and 18. At the GTG-band level the karyotype was abnormal in 20% of the analyzed cells. The complex karyotype was studied in more detail by spectral karyotyping (SKY) and multicolor banding (MCB) to characterize it in more detail. Thus, the karyotype could be described very accurately and in summary three different clones were detected, reflecting a high rate of karyotypic evolution in this patient.     

Multicolor banding technique, spectral color banding (SCAN): new development and applications

Conventional banding techniques can characterize chromosomal aberrations associated with tumors and congenital diseases with considerable precision. However, chromosomal aberrations that have been overlooked or are difficult to analyze even by skilled cytogeneticists were also often noted. Following the introduction of multicolor karyotyping such as spectral karyotyping (SKY) and multiplex-fluorescence in situ hybridization (M-FISH), it is possible to identify this kind of cryptic or complex aberration comprehensively by a single analysis. To date, multicolor karyotyping techniques have been established as useful tools for cytogenetic analysis. However, since this technique depends on whole chromosome painting probes, it involves limitations in that the origin of aberrant segments can be identified only in units of chromosomes. To overcome these limitations, we have recently developed spectral color banding (SCAN) as a new multicolor banding technique based on the SKY methodology. This new technique may be deemed as an ideal chromosome banding technique since it allows representation of a multicolor banding pattern matching the corresponding G-banding pattern. We applied this technique to the analysis of chromosomal aberrations in tumors that had not been fully characterized by G-banding or SKY and found it capable of (1) detecting intrachromosomal aberrations; (2) identifying the origin of aberrant segments in units of bands; and (3) precisely determining the breakpoints of complex rearrangements. We also demonstrated that SCAN is expected to allow cytogenetic analysis with a constant adequate resolution close to the 400-band level regardless of the degree of chromosome condensation. As compared to the conventional SKY analysis, SCAN has remarkably higher accuracy for a particular chromosome, allowing analysis in units of bands instead of in units of chromosomes and is hence promising as a means of cytogenetic analysis.     

Rapid physical mapping of cloned DNA on banded mouse chromosomes by fluorescence in situ hybridization.

Physical mapping of DNA clones by nonisotopic in situ hybridization has greatly facilitated the human genome mapping effort. Here we combine a variety of in situ hybridization techniques that make the physical mapping of DNA clones to mouse chromosomes much easier. Hybridization of probes containing the mouse long interspersed repetitive element to metaphase chromosomes produces a Giemsa-like banding pattern which can be used to identify individual Mus musculus, Mus spretus, and Mus castaneus chromosomes. The DNA binding fluorophore, DAPI, gives quinacrine-like bands that can complement the hybridization banding data. Simultaneous hybridization of a differentially labeled clone of interest with the banding probe allows the assignment of a mouse clone to a specific cytogenetic band. These methods were validated by first mapping four known genes, Cpa, Ly-2, Cck, and Igh-6, on banded chromosomes. Twenty-seven additional clones, including twenty anonymous cosmids, were then mapped in a similar fashion. Known marker clones and fractional length measurements can also provide information about chromosome assignment and clone order without the necessity of recognizing banding patterns. Clones hybridizing to each murine chromosome have been identified, thus providing a panel of marker probes to assist in chromosome identification.     

Spectral karyotyping analysis of human and mouse chromosomes

Classical banding methods provide basic information about the identities and structures of chromosomes on the basis of their unique banding patterns. Spectral karyotyping (SKY), and the related multiplex fluorescence in situ hybridization (M-FISH), are chromosome-specific multicolor FISH techniques that augment cytogenetic evaluations of malignant disease by providing additional information and improved characterization of aberrant chromosomes that contain DNA sequences not identifiable using conventional banding methods. SKY is based on cohybridization of combinatorially labeled chromosome-painting probes with unique fluorochrome signatures onto human or mouse metaphase chromosome preparations. Image acquisition and analysis use a specialized imaging system, combining Sagnac interferometer and CCD camera images to reconstruct spectral information at each pixel. Here we present a protocol for SKY analysis using commercially available SkyPaint probes, including procedures for metaphase chromosome preparation, slide pretreatment and probe hybridization and detection. SKY analysis requires approximately 6 d.     

A photographic representation of the variability in the G-banded structure of the chromosomes in the mouse karyotype

Analysis of the mouse chromosomes is becoming increasingly important in many fields of genetic research. It is generally considered that the mouse chromosomes are more difficult to analyse than, for example, human chromosomes which has often led to their misidentification. This article presents a guide to the correct identification of trypsin-Giemsa banded chromosomes from the mouse. The variability in the G-banded structure of each chromosome is presented pictorially together with some suggestions for their unequivocal identification. Since many of the mouse chromosomes have similar banding patterns, those chromosomes which are more frequently misidentified have been compared and contrasted. Finally a summary of the main features for the identification of each chromosome is presented.     

A method for analysis of chromosomes in hybrid cells employing sequential G-banding and mouse specific C-banding

A sequential banding technique is described for the identification of chromosomes of interspecific hybrid cells with a mouse parent. Metaphases were first G-banded using trypsin-Giemsa to identify individual chromosomes and then the centromeres of the same cells were differentially stained by a C-banding technique specific for mouse chromosomes. This mouse specific C-banding employs treatment with hot formamide-SSC before staining, and the effect of this treatment on the staining of chromosomes from a number of species was investigated. The specific staining of mouse centromeres confirms the parental origin of chromosomes identified by G-banding and allows the rapid recognition of mouse and non-mouse chromosomes in metaphases from many different hybrid combinations.     

Evolutionary relationships between rat and mouse chromosomes

Trypsin banded karyotypes of rat and mouse chromosomes were analyzed for banding pattern similarities. Apparently identical banding patterns were found, covering about 40% of the genome of each species. Predictions are made as to what gene loci are located on specific rat chromosomes. The problem of the relationship between the genetic content of a chromosome and its banding pattern is discussed.     

The karyotype of the mouse

A denaturating and renaturating technique, applied to mouse chromosomes, makes visible characteristic banding patterns by which all elements of the karyotype can be individually distinguished. The Y chromosome as a whole appears darkly stained. The X chromosome comprises 6.33% of the homogametic haploid set. The banding pattern of the chromosomes is compared with that obtained by aid of the quinacrine dihydrochloride fluorescence technique. After its use a banding pattern results which is similar to, but less distinct than, that found after the renaturation procedure.     

Learning to karyotype in the university environment: a computer-based virtual laboratory class (KaryoLab) designed to rationalize time for the tutor/researcher and to encourage more students to engage in cytogenetics

The ability to karyotype G-banded chromosome preparations is an essential skill for chromosome biologists. For this reason, the teaching of the rudiments of G banding analysis forms an integral part of the curriculum in many biology and genetics degree courses. The way in which karyotyping is usually taught involves providing the students with a photograph of G-banded chromosomes, a pair of scissors and some glue from which they can cut out the chromosomes and build the karyotype. This has the disadvantage that large amounts of time are taken in cutting and pasting and comparatively little in learning pattern recognition of individual chromosomes. In this paper we describe the development of a computer-based student practical class "KaryoLab". To the best of our knowledge, this is the first report of a teaching tool that combines instruction in cytogenetic analysis with both formative and summative feedback to the student and a virtual elimination of marking time for the tutor. Chromosome research and diagnostics will only continue while there are sufficiently motivated and trained individuals to perform it. We see the software developed here as a significant step towards training and motivating students in cytogenetics.     

Cloning and Characterization of Chromosomal Markers from a Cot-1 Library of Peanut ( Arachis hypogaea L.)

The cultivated peanut, Arachis hypogaea (AABB, 2n = 40), is an allotetraploid which was probably originated from a hybridization event between 2 ancestors, A. duranensis (A genome) and A. ipaensis (B genome) followed by chromosome doubling. The wild species in the Arachis section are useful genetic resources for genes that confer biotic and abiotic stress resistance for peanut breeding. However, the resource is not well exploited because little information on the genetic, cytogenetic, and phylogenetic relationships between cultivated peanut and its wild relatives is known. Characterization of its chromosome components will benefit the understanding of these issues. But the paucity of information on the DNA sequence and the presence of morphologically similar chromosomes impede the construction of a detailed karyotype for peanut chromosome identification. In our study, a peanut Cot-1 library was constructed to isolate highly and moderately repetitive sequences from the cultivated peanut, and the chromosomal distributions of these repeats were investigated. Both genome and chromosome specific markers were identified that allowed the distinguishing of A and B genomes in tetraploid peanut and a possible karyotyping of peanut chromosomes by FISH. In particular, a 115-bp tandem repetitive sequence was identified to be a possible centromere repetitive DNA, mainly localized in the centromeres of B chromosomes, and a partial retrotransposable element was also identified in the centromeres of B chromosomes. The cloning and characterization of various chromosomal markers is a major step for FISH-based karyotyping of peanut. The FISH markers are expected to provide a reference tool for sequence assembly, phylogenetic studies of peanut and its wild species, and breeding.     

Computer assisted karyotype analysis of Pyrrhopappus carolinianus

With the help of Computer Aided Karyotyping procedures, Ag-NOR staining and C-banding techniques, the karyotype of Pyrrhopappus carolinianus (Asteraceae, Lactuceae) has been studied. The species has 2n=12 chromosomes. Silver staining reveals that the two shortest pairs of chromosomes possess NOR's. On the basis of chromosome length and centromere position, only the longest chromosome pair and the satellite chromosomes can be identified. Two types of C-banding can be obtained, dependent on the temperature of the hydrochloric acid hydrolysis of the root tips. Hydrolysis at 60°C results exclusively in centromeric bands, whereas a treatment at room temperature reveals a pattern of intercalary bands. A computer assisted analysis of the intercalary banding pattern resulted in the construction of schematic representation of the average C-banding pattern. This banding pattern allows an easy identification of each of the chromosome pairs.     

AcroM fluorescent in situ hybridization analyses of marker chromosomes

The presence of a de novo supernumerary marker chromosome (SMC) poses problems in genetic counseling. The consequences of the additional chromosomal material may range from harmless to detrimental. As the composition of a SMC cannot be deciphered by traditional banding analysis, sophisticated methods are needed for their rapid and detailed analyses. A new strategy is presented, which allows the elucidation of the composition of SMCs in one or two hybridizations. One hybridization, termed AcroM-FISH, involves a newly generated probe mix, which consists of painting probes for all acrocentric chromosomes, centromere probes for chromosomes 13/21, 14/22, 15, and a probe specific for rDNA, each labeled with a specific combination of fluorochromes. This probe mix is sufficient to characterize approximately 80% of all SMCs. For the other 20% of SMCs, chromosomes can be analyzed in a second hybridization by multicolor karyotyping, for example, multiplex FISH (M-FISH), to check for the presence of euchromatin of other chromosomes. The potential of AcroM-FISH was tested in various applications.