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.     

多色-FISH技术的进展及其在分子生物医学上的应用

传统显带分析技术以每条染色体独特的显带带型为依据,提供染色体形态结构的基本信息,用于染色体核型的初步分析。然而有些染色体重排由于涉及的片断太小或具有相似的带型,用该方法难以探测或准确描绘。多元荧光原位杂交(M-FISH),光谱核型分析(SKY),FISH-显带分析技术是染色体特异的多色荧光原位杂交技术(mFISH)。它们能够探测出传统显带分析不能发现的染色体异常,提供更准确的核型。M-FISH和SKY均以组合标记的染色体涂染探针共杂交为基础,二者的不同在于观察仪器和分析方法上。它们可对中期染色体涂片进行快速准确分析,描绘复杂核型,确认标记染色体,主要用于恶性疾病的细胞遗传学诊断分析。FISH-显带分析技术以FISH技术为基础,能同时检测多条比染色体臂短的染色体亚区域。符合该定义的FISH-显带分析技术各有特点,其共同特点是都能产生DNA特异的染色体条带。这些条带有更多色彩,能提供更多信息。FISH-显带分析技术已经成功地被用于进化生物学、放射生物学以及核结构的研究,同时也被用于产前、产后以及肿瘤细胞遗传学诊断,是很有潜力的工具。     

Multicolor spectral karyotyping of rat chromosomes

Rat and mouse have become important animal models to study various human diseases such as cancer. Cytogenetic analysis of the respective karyotypes is frequently required to investigate the causative genetic defects and especially neoplastic cells often show complex chromosome aberrations and many different marker chromosomes. However, structural homogeneity of the chromosomes in these species as well as less pronounced differences in banding patterns make it difficult to assign genetic abnormalities to certain chromosomes by conventional banding techniques. Here we report for the first time the successful application of multicolor spectral karyotyping (SKY) to rat chromosomes, which allows unequivocal identification of all rat chromosomes with the exception of chromosomes 13 and 14 in different colors, thus enabling the elucidation of even complex rearrangements in the rat karyotype. Flow-sorted chromosome specific painting probes for all 22 rat chromosomes (20 autosomes, X, and Y) were combinatorially labeled by a set of five different fluorochromes and hybridized in situ to metaphase spreads of a healthy rat, to diakineses from testicular material, and to cells from a rat FAO hepatoma cell line. Measuring the complete spectrum at each image point by using the SpectraCube((R)) spectral imaging system and respective computer software allowed identification of the individual rat chromosomes by their specific emission spectra. Classification algorithms in the analysis software can then display the rat chromosomes in specific pseudo-colors and automatically order them in a karyotype table. After its successful application to human and mouse chromosomes, spectral karyotyping of rat chromosomes now also allows cytogenetic screening of the complete rat genome by a single hybridization.     

Identification of de novo chromosomal markers and derivatives by spectral karyotyping

Despite major advances in molecular cytogenetics during the past decade and the important diagnostic role that fluorescence in situ hybridization (FISH) plays in the characterization of chromosomal abnormalities, the usefulness of this technique remains limited by the number of spectrally distinguishable fluorochromes or fluorochrome combinations. Overcoming this major limitation would allow one to use FISH to screen the whole human genome for chromosomal aberrations which, until recently, was possible only through conventional karyotyping. A recently described molecular cytogenetics technology, 24-color FISH using spectral karyotyping (SKY), permits the simultaneous visualization of all human chromosomes in 24 different colors. Most chromosomal aberrations detected during cytogenetic evaluation can be resolved using routine cytogenetic techniques alone or in combination with single- or dual-color FISH. However, some cases remain unresolved, in particular de novo supernumerary marker chromosomes and de novo unbalanced structural rearrangements. These findings cause particular diagnostic and counseling concerns when detected during prenatal diagnosis. The purpose of this report is to demonstrate the application of SKY in the characterization of these de novo structural chromosomal abnormalities. Eight cases are described in this report. SKY has considerable diagnostic applications in prenatal diagnosis because of its reliability and speed. The identification of the chromosomal origin of markers and unbalanced translocations provides the patient, physician, and genetic counselor with better predictive information on the phenotype of the carrier. Received: 2 June 1998 / Accepted: 16 June 1998     

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     

Identification and fate of a marker chromosome in methotrexate-resistant V79,B7 cells by flow karyotyping and sorting, metaphase analysis and in situ hybridization.

The chromosomes from a methotrexate (MTX)-resistant and its parental V79,B7 Chinese hamster cell line were analysed by the combined use of flow karyotyping and sorting, metaphase analysis and in situ hybridization with a probe for the dihydrofolate reductase (DHFR) gene responsible for methotrexate resistance. A marker chromosome with an elongated arm carrying the amplified DHFR gene was identified by in situ hybridization of metaphase cells of the methotrexate-resistant line. In the flow karyotype the marker chromosome was found as an additional peak with a higher DNA content compared with the largest chromosome of the sensitive line. This was additionally verified by G-banding of the chromosomes sorted from the marker peak. Several other chromosomal rearrangements not associated with the amplified gene could be identified in the methotrexate-resistant line by the combined use of flow karyotyping and metaphase analysis. The fate of the original marker chromosome was studied in cells growing several weeks in the absence of methotrexate, comparing flow karyotyping and metaphase analysis. The original marker chromosome was lost in about 50% of the cells after 5 weeks and in about 60% of the cells after 8 weeks; between 80 and 90% of the cells, however, contained marker chromosomes of various sizes. The MTX-resistance decreased in parallel during loss of the original marker chromosome. In conclusion, the study shows that the power of cytogenetic analysis is improved by the combined use of conventional cytogenetics, molecular cytogenetics and flow cytometry.     

Comparison between the G-banded karyotype of the aoudad (Ammotragus lervia) and sheep (Ovis aries)

Karyotypes of the aoudad and sheep were compared on the basis of G-banded chromosomes at the 450 band level. The common G-banded karyotype showed the homology of all aoudad chromosomes (2n=58) with sheep chromosomes (2n=54) or sheep chromosome arms. The results of cytogenetic investigations suggest that in this case karyotype evolution has led to reduction in chromosome number as a result of centric fusions. The formation of the first metacentric chromosome occurred in the aoudad. The homology of the G-banding pattern in sheep and aoudad suggests the conservation in linear arrangement of genetic material. Thus comparative cytogenetics can be a useful tool in gene mapping.     

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).     

Delineation of marker chromosomes by reverse chromosome painting using only a small number of DOP-PCR amplified microdissected chromosomes

A new procedure for determining the chromosomal origin of marker chromosomes has been carried out. The origin of marker chromosomes that were unidentifiable by standard banding techniques could be verified by reverse chromosome painting. This technique includes microdissection, followed by in vitro DNA amplification and fluorescence in situ hybridization (FISH). A number of marker chromosomes prepared from unbanded and from GTG-banded lymphocyte chromosomes were collected with microneedles and transferred to a collection drop. The chromosomal material was amplified by a degenerate oligonucleotide-primed polymerase chain reaction (DOP-PCR). The resulting PCR products were labelled by nick-translation with biotin-11-dUTP and used as probes for FISH. They were hybridized onto normal metaphase spreads in order to determine the precise regional chromosomal origin of the markers. Following this approach, we tested 2–14 marker chromosomes in order to determine how many are necessary for reverse chromosome painting. As few as two marker chromosomes provided sufficient material to paint the appropriate chromosome of origin, regardless of whether the marker contained heterochromatic or mainly euchromatic material. With this method, it was possible to identify two marker chromosomes of a healthy proband [karyotype: 48,XY, +mar₁,+mar₂] and an aberrant Y chromosome of a mentally retarded boy [karyotype: 46,X, der(Y)].     

A boy with small supernumerary marker chromosome X identified by FISH

Marker or ring X chromosomes are frequently seen in Ullrich-Turner Syndrome with 46,X,r(X) karyotype, but only 8 children were reported with an extra marker X chromosome in at least some of their cell lines, we describe a 5 years old male patient who is mosaic (17%) for a cell line with an extra ring shaped marker X chromosome in addition to a normal 46,XY cell line. He had mild motor mental retardation, a dysmorphic face, dysplastic ears, high arched palate, cryptorchidism and brachydactyly. G-banding showed 46,XY[83]/47,XY,+r?[17] karyotype. NOR banding revealed no satellite region but its centromere was intact in C-banding. By fluorescent in situ hybridization (FISH) technique, dual X/Y alpha-satellite probes were used to detect the origin of ring shaped marker chromosome and 17% of his cells had two X chromosome signals due to marker X; hybridization with X chromosome inactivation center (XIST) specific probe revealed the absence of the locus on the ring chromosome. In this report, clinical features of our patient are compared with previously reported cases and the cytogenetic and molecular cytogenetic techniques used to detect origin of marker chromosome are discussed.     

Chromosomal phylogeny and evolution of gibbons (Hylobatidae)

Although human and gibbons are classified in the same primate superfamily (Hominoidae), their karyotypes differ by extensive chromosome reshuffling. To date, there is still limited understanding of the events that shaped extant gibbon karyotypes. Further, the phylogeny and evolution of the twelve or more extant gibbon species (lesser apes, Hylobatidae) is poorly understood, and conflicting phylogenies have been published. We present a comprehensive analysis of gibbon chromosome rearrangements and a phylogenetic reconstruction of the four recognized subgenera based on molecular cytogenetics data. We have used two different approaches to interpret our data: (1) a cladistic reconstruction based on the identification of ancestral versus derived chromosome forms observed in extant gibbon species; (2) an approach in which adjacent homologous segments that have been changed by translocations and intra-chromosomal rearrangements are treated as discrete characters in a parsimony analysis (PAUP). The orangutan serves as an "outgroup", since it has a karyotype that is supposed to be most similar to the ancestral form of all humans and apes. Both approaches place the subgenus Bunopithecus as the most basal group of the Hylobatidae, followed by Hylobates, with Symphalangus and Nomascus as the last to diverge. Since most chromosome rearrangements observed in gibbons are either ancestral to all four subgenera or specific for individual species and only a few common derived rearrangements at subsequent branching points have been recorded, all extant gibbons may have diverged within relatively short evolutionary time. In general, chromosomal rearrangements produce changes that should be considered as unique landmarks at the divergence nodes. Thus, molecular cytogenetics could be an important tool to elucidate phylogenies in other species in which speciation may have occurred over very short evolutionary time with not enough genetic (DNA sequence) and other biological divergence to be picked up.Electronic Supplementary Material Supplementary material is available in the online version of this article at     

Combined RxFISH/G-banding allows refined karyotyping of solid tumors

Chromosome banding analysis of solid tumors often yields incomplete karyotypes because of the complex rearrangements encountered. The addition of fluorescence in situ hybridization (FISH) methods has helped improve the accuracy of solid tumor cytogenetics, but the absence of screening qualities from standard FISH approaches has proved a severe limitation. We describe the cytogenetic analysis of ten solid tumors using G-banding followed by cross-species color banding (RxFISH), a FISH-based screening technique giving a chromosome-specific banding pattern based on the genomic homologies between humans and gibbons. The addition of RxFISH analysis in all cases led to the identification of previously unidentified intra- as well as interchromosomal rearrangements, thus giving a much more certain and detailed karyotype. In two gastric stromal sarcomas, a tumor type for which no cytogenetic data were hitherto available, numerical chromosomal aberrations dominated, but one of the tumors also carried an unbalanced 7;17-translocation with the same breakpoint in chromosome 17 as that seen in endometrial stromal sarcomas. Received: 15 January 1999 / Accepted: 5 March 1999     

Chromosomal rearrangements in cattle and pigs revealed by chromosome microdissection and chromosome painting

A pericentric inversion of chromosome 4 in a boar, as well as a case of (2q-;5p+) translocation mosaicism in a bull were analysed by chromosome painting using probes generated by conventional microdissection. For the porcine inversion, probes specific for p arms and q arms were produced and hybridised simultaneously on metaphases of a heterozygote carrier. In the case of the bovine translocation, two whole chromosome probes (chromosome 5, and derived chromosome 5) were elaborated and hybridised independently on chromosomal preparations of the bull who was a carrier of the mosaic translocation. The impossibility of differentiating chromosomes 2 and der(2) from other chromosomes of the metaphases did not allow the production of painting probes for these chromosomes. For all experiments, the quality of painting was comparable to that usually observed with probes obtained from flow-sorted chromosomes. The results obtained allowed confirmation of the interpretations proposed with G-banding karyotype analyses. In the bovine case, however, the reciprocity of the translocation could not be proven. The results presented in this paper show the usefulness of the microdissection technique for characterising chromosomal rearrangements in species for which commercial probes are not available. They also confirmed that the main limiting factor of the technique is the quality of the chromosomal preparations, which does not allow the identification of target chromosomes or chromosome fragments in all cases.     

Classical and molecular cytogenetics of the zebrafish, Danio rerio (Cyprinidae, Cypriniformes): an overview

The zebrafish, Danio rerio, has recently become the model system for the genetic analysis of vertebrate development. This paper reviews the advances in zebrafish cytogenetics, obtained through classical and molecular techniques, which will lead to the assignment of specific linkage groups to specific chromosome pairs in the zebrafish genome project. Several chromosome pairs of the 50-chromosome karyotype of D. rerio were differentially stained by classical staining techniques and additional information has been obtained by molecular cytogenetics. Indeed, the analysis of constitutive heterochromatin by C-banding and base-specific fluorochrome staining had suggested a differential composition of peri- and paracentromeric constitutive heterochromatin. The chromosome mapping of distinct AT- and GC-rich zebrafish satellite DNAs by means of PRINS (Primed in situ) and multicolor FISH (Fluorescence in situ Hybridization) has confirmed this hypothesis, which therefore provided the chromosome localization of 10% of the zebrafish genome. The analysis of nucleolus organizer regions (NORs) by silver staining and by FISH with 18S rDNA has also revealed the existence of variable and inactive NORs, in addition to those on the terminal regions of the long arms of the three NOR-bearing chromosome pairs. Other multicopy genes, such as minor ribosomal genes, or multicopy repeats, such as telomere specific sequences, have now been mapped on zebrafish chromosomes. The latest advancement in zebrafish molecular cytogenetics is the chromosome mapping of single locus genes. Single-copy genes from each of the 25 genetic linkage groups are now being mapped on zebrafish chromosomes by using PAC clones.     

大麦G—显带核型的研究

本文报道了 ASG 法处理的三个栽培大麦(Hordeum Vulgare)品种 G-带的核型研究。结果表明无论是早中期或中期染色体都显示出了密切邻近的、多重的 G-带带纹。在有丝分裂过程中染色体愈浓缩带纹数目愈少。同源染色体之间带纹分布的位置、染色深浅以及带纹数目都基本一致,可以较为准确地进行配对。同一分裂时期不同染色体的 G-带带纹各具一定的特点,可以作为鉴别的标记。讨论了显带技术和中期染色体的 G-带等问题。     

Studies on G-Banding Karyotype in Barley (Hordeum vulgare)

G-banding karyotypes of three cultivars in barley were analyzed. Multiple closely adjacent G-bands were able to be observed in each early metaphase or metaphase chromosome treatted by an ASG method. The more concentrated the chromosome, the less was the number of G-bands during mitosis. The position of band distribution, staining degree and band numbers between homologous chromosomes were basically identical. Chromosome pairing for karyotype analysis could be carried out more accurately. G-banding patterns of different chromosome pairs were not the same, they could be used as the markers to distinguish one from another chromosome pair. During the same mitotic stage the banding patterns including number, relative position and staining degree of the bands between different cultivars were basically the same, but they had differences in the size and staining degree of some bands near centromeres. G-banding technique and G-banding of metaphase chromosomes were discussed.     

Chromosome evolution in new world monkeys (platyrrhini)

During the last decades, New World monkey (NWM, Platyrrhini, Anthropoideae) comparative cytogenetics has shed light on many fundamental aspects of genome organisation and evolution in this fascinating, but also highly endangered group of neotropical primates. In this review, we first provide an overview about the evolutionary origin of the inferred ancestral NWM karyotype of 2n = 54 chromosomes and about the lineage-specific chromosome rearrangements resulting in the highly divergent karyotypes of extant NWM species, ranging from 2n = 16 in a titi monkey to 2n = 62 in a woolly monkey. Next, we discuss the available data on the chromosome phylogeny of NWM in the context of recent molecular phylogenetic analyses. In the last part, we highlight some recent research on the molecular mechanisms responsible for the large-scale evolutionary genomic changes in platyrrhine monkeys.     

Clonal derivatives of a human B-lymphoblastoid cell line producing prolactin—A cytogenetic characterization

The IM-9-P cell line is a variant of the human B-lymphoblastoid cell line IM-9 which ectopically secretes prolactin (hPRL). The heterogeneous line IM-9-P and three sublines of clonal origin, two of them positive and one negative for PRL gene expression, were subjected to cytogenetic analysis and compared with the reference line IM-9 which showed a normal female diploid karyotype. G-banding revealed several rearrangements in the chromosomes. Nine altered chromosomes including one stable marker chromosome were common to all analysed karyotypes of IM-9-P cells and their clones. A second marker chromosome mar2 occurred only in the karyotypes of the hPRL producing clones, but not in the non-producing clone. None of the visible alterations involve chromosome 6 which carries the PRL gene in humans.     

Toward a multicolor chromosome bar code for the entire human karyotype by fluorescence in situ hybridization

A colored banding pattern for human chromosomes is described that distinguishes each chromosome in a single fluorescence in situ hybridization with a set of subregional DNA probes. Alu/polymerase chain reaction products of various human/rodent somatic cell hybrids (fragment hybrids) were pooled into two probe sets that were labeled differentially and detected by red and green fluorescence. Chromosome regions hybridized by DNA present in both pools appeared yellow. The result was a multi-color set of 110 distinct signals per haploid chromosome set for the human karyotype. Each individual chromosome showed a unique sequence of signals, a result termed the “chromosome bar code”. The reproducibility of the hybridization pattern in various labeling and hybridization experiments was analyzed by computer densitometry. We have applied the chromosome bar code both in diagnostic cytogenetics and in genome studies. The approach allows the rapid identification of chromosomes and chromosome rearrangements. Although not yet showing the resolution of classical banding patterns, the present experiments demonstrate various applications in which the present multi-color bar code can significantly add to the spectrum of cytogenetic techniques. Received: 18 December 1996 / Accepted: 10 February 1997