Chromosome Analysis Using Spectral Karyotyping (SKY)

Spectral karyotyping is a novel technique for chromosome analysis that has been developed based on the approach of the fluorescence in situ hybridization technique. Spectral karyotyping makes it feasible to diagnose a variety of diseases, because of its technology in painting each of the 24 human chromosomes with different colors. In recent years, it has become possible to adopt the usage of spectral karyotyping for research in general clinical practice, and its usability has attracted particular attention in the diagnosis of different diseases. In this review, we will explain the principle of the spectral karyotyping, as well as its specificity and limitation in detecting the genetic defects within clinical application by presenting two case reports.     

Karyotyping Human Chromosomes by Optical and X-Ray Ptychography Methods

Sorting and identifying chromosomes, a process known as karyotyping, is widely used to detect changes in chromosome shapes and gene positions. In a karyotype the chromosomes are identified by their size and therefore this process can be performed by measuring macroscopic structural variables. Chromosomes contain a specific number of basepairs that linearly correlate with their size; therefore, it is possible to perform a karyotype on chromosomes using their mass as an identifying factor. Here, we obtain the first images, to our knowledge, of chromosomes using the novel imaging method of ptychography. We can use the images to measure the mass of chromosomes and perform a partial karyotype from the results. We also obtain high spatial resolution using this technique with synchrotron source x-rays.     

Single-gene detection and karyotyping using small-target fluorescence in situ hybridization on maize somatic chromosomes

Combined with a system for identifying each of the chromosomes in a genome, visualizing the location of individual genetic loci by fluorescence in situ hybridization (FISH) would aid in assembling physical and genetic maps. Previously, large genomic clones have been successfully used as FISH probes onto somatic chromosomes but this approach is complicated in species with abundant repetitive elements. In this study, repeat-free portions of sequences that were anchored to particular chromosomes including genes, gene clusters, large cDNAs, and portions of BACs obtained from public databases were used to label the corresponding physical location using FISH. A collection of probes that includes at least one marker on each chromosome in the maize complement was assembled, allowing a small-target karyotyping system to be developed. This set provides the foundation onto which additional loci could be added to strengthen further the ability to perform chromosomal identification in maize and its relatives. The probes were demonstrated to produce signals in several wild relatives of maize, including Zea luxurians, Z. diploperennis, and Tripsacum dactyloides.     

A new multicolor-FISH approach for the characterization of marker chromosomes: centromere-specific multicolor-FISH (cenM-FISH)

Centromere-specific multi-color FISH (cenM-FISH) is a new multicolor FISH technique that allows the simultaneous characterization of all human centromeres by using labeled centromeric satellite DNA as probes. This approach allows the rapid identification of all human centromeres by their individual pseudo-coloring in one single step and is therefore a powerful tool in molecular cytogenetics. CenM-FISH fills a gap in multicolor karyotyping using WCP probes and distinguishes all centromeric regions apart from the evolutionary highly conserved regions on the chromosomes 13 and 21. The usefulness of the cenM-FISH technique for the characterization of small supernumerary marker chromosomes with no (or nearly no) euchromatin and restricted amounts of available sample material is demonstrated in prenatal, postnatal, and tumor cytogenetic cases. In addition, rarely described markers with the involvement of heterochromatic material inserted into homogeneously staining regions could be identified and characterized by using the cenM-FISH technique.     

Current status and the future of fluorescence in situ hybridization (FISH) in plant genome research.

Fluorescence in situ hybridization (FISH), which allows direct mapping of DNA sequences on chromosomes, has become the most important technique in plant molecular cytogenetics research. Repetitive DNA sequence can generate unique FISH patterns on individual chromosomes for karyotyping and phylogenetic analysis. FISH on meiotic pachytene chromosomes coupled with digital imaging systems has become an efficient method to develop physical maps in plant species. FISH on extended DNA fibers provides a high-resolution mapping approach to analyze large DNA molecules and to characterize large genomic loci. FISH-based physical mapping provides a valuable complementary approach in genome sequencing and map-based cloning research. We expect that FISH will continue to play an important role in relating DNA sequence information to chromosome biology. FISH coupled with immunoassays will be increasingly used to study features of chromatin at the cytological level that control expression and regulation of genes.     

多重荧光定量PCR技术快速诊断21三体及18三体方法的建立及临床应用

利用收集的20例21三体、3例18三体DNA样本及40例正常人DNA样本,选择多对21、18号染色体短串联重复序列分子标记,建立多重荧光定量PCR检测技术用于21三体、18三体的快速产前诊断;利用建立的方法对165例产前诊断病例及4例消化道畸形新生儿进行检测,并与核型分析结果相比较。169例病例中共诊断21三体4例,18三体1例,所有病例均在1～3d得到结果,无漏诊和误诊,并利用建立的多重荧光定量PCR技术为5例核型分析失败的病例提供了明确的产前诊断。对于同期B超检查胎儿结构异常的22例胎儿,则采用传统的核型分析,检出1例(45,X),1例(47,XXY)。结果表明建立的多重荧光定量PCR技术可快速、准确地诊断21三体和18三体,减轻核型分析需时过长给孕妇带来的焦虑,可用于血清学筛查21三体、18三体高风险者及高龄孕妇,也适用于对其他遗传性疾病如遗传性耳聋进行产前诊断时并行检测以排除21三体和18三体。多重荧光定量PCR技术结合传统核型分析可更好的满足产前诊断的临床需求。     

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     

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.     

High-resolution chromosomes of rhesus macaques (Macaca mulatta)

Late-prophase high-resolution chromosomes were successfully cultured for 22 of 27 Macaca mulatta samples. Twelve of the successful cultures were adequate for karyotyping high-resolution spreads. High-resolution chromosome technique provides an important contribution to primate genetics because it can be used to identify chromosomal anomalies undetected in metaphase spreads and may be useful for paternity exclusion analysis.     

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.     

Chromosomes in the flow to simplify genome analysis

Nuclear genomes of human, animals, and plants are organized into subunits called chromosomes. When isolated into aqueous suspension, mitotic chromosomes can be classified using flow cytometry according to light scatter and fluorescence parameters. Chromosomes of interest can be purified by flow sorting if they can be resolved from other chromosomes in a karyotype. The analysis and sorting are carried out at rates of 10(2)-10(4) chromosomes per second, and for complex genomes such as wheat the flow sorting technology has been ground-breaking in reducing genome complexity for genome sequencing. The high sample rate provides an attractive approach for karyotype analysis (flow karyotyping) and the purification of chromosomes in large numbers. In characterizing the chromosome complement of an organism, the high number that can be studied using flow cytometry allows for a statistically accurate analysis. Chromosome sorting plays a particularly important role in the analysis of nuclear genome structure and the analysis of particular and aberrant chromosomes. Other attractive but not well-explored features include the analysis of chromosomal proteins, chromosome ultrastructure, and high-resolution mapping using FISH. Recent results demonstrate that chromosome flow sorting can be coupled seamlessly with DNA array and next-generation sequencing technologies for high-throughput analyses. The main advantages are targeting the analysis to a genome region of interest and a significant reduction in sample complexity. As flow sorters can also sort single copies of chromosomes, shotgun sequencing DNA amplified from them enables the production of haplotype-resolved genome sequences. This review explains the principles of flow cytometric chromosome analysis and sorting (flow cytogenetics), discusses the major uses of this technology in genome analysis, and outlines future directions.     

Karyotyping of Brassica amphidiploids using 5S and 25S rDNA as chromosome markers

Species of Brassica have small, morphologically similar chromosomes, which makes karyotyping difficult using conventional cytogenetic methods. Molecular cytogenetic methods, like fluorescence in situ hybridisation (FISH) have the potential to improve karyotyping through the use of chromosome- or genome-specific markers. Simultaneous application of more than one DNA probe can greatly enrich the results obtained compared with separate single target FISH experiments. This paper demonstrates the use of multicolour fluorescence in situ hybridisation with 5S and 25S rDNA for karyotyping three amphidiploid species: B. napus, B. juncea and B. carinata. Using this method, it was possible to identify eight out of nineteen pairs of chromosomes in B. napus, ten out of eighteen pairs in B. juncea and six out of sixteen pairs in B. carinata. Additionally, rDNA sites allow the determination of the genomic origin of all marked chromosomes in B. napus and B. juncea.     

Spectral karyotyping (SKY) of mouse meiotic chromosomes.

The spectral karyotyping procedure of in situ hybridization with chromosome-specific probes assigns a unique colour code to each of the 21 mouse mitotic chromosomes. We have adapted this procedure to meiotic prophase chromosomes, and the results show that each of the pachytene or metaphase I bivalents can be identified. This technique has the potential to recognize synaptic anomalies and chromosome-specific structural and behavioural characteristics. We confirm these potentials by the recognition of the heterologous synapsis of the X and Y chromosomes and by the variances of synaptonemal complex lengths for each of the colour-coded bivalents in eight prophase nuclei.     

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     

Investigation of karyotypic composition and evolution in <Emphasis Type="Italic">Lilium</Emphasis> species belonging to the section martagon

Analyzing chromosomal traits is one of the pragmatic ways to establish evolutionary and genetic database of plants that has complicated phylogenetic system. There are some conflicts on the exact phylogeny and evolutionary pathway of Lilium, and section martagon is the most complicated part among them. In this study, chromosomal traits of martagon lily species are described. All martagon lilies were analyzed with FISH (Fluorescence in situ hybridization) technique, followed by detailed karyotyping. Each species showed 2n = 2x = 24 of chromosome complement. Size of chromosomes ranged from 451.04 to 680.06 µm. 5S and 45S ribosomal DNA, general molecular markers in modern evolutionary research were used as probe in this study. Variation in rDNA loci and chromosome translocation were observed in Lilium hansonii; the highest number of 45S rDNA loci was detected in Lilium hansonii, followed by other martagon lilies, in similar locations but with differences, and chromosome translocation was observed from one individual of Lilium hansonii. Additionally, Lilium tsingtauense from Jeju-do Island, Korea was detected with two extra chromosomes. These kind of genetic variations through karyotyping indicate ongoing genetic variations in martagon lilies. In this study, precise analysis of chromosome traits in Lilium species belonging to section martagonperformed to contribute to better comprehension of the evolutionary pathway and establishment of cytogenetic database for further plant breeding research.     

The Athena semi-automated karyotyping system

In this article we describe Athena, a system that provides for semi-automated karyotyping of metaphase spreads. The system is based upon the Macintosh II computer. It uses software that is written entirely in C and consists of approximately 200 Kbytes of executable code. Athena provides automated segmentation of metaphase images into individual chromosomes, automated measurements on each banded chromosome, and automated classification into the standard Paris-convention karyotype. Furthermore, the system provides the ability to construct one or more chromosome data bases to represent the types of metaphase spreads and staining techniques that may be used in a given laboratory. Because we believe that it is impossible to construct a system that can achieve perfect segmentation, perfect separation of touching and overlapping chromosomes, perfect localization of the centromeres, and perfect classification, the system offers the possibility for interaction at each of the above stages using the well-accepted Macintosh user interface.     

Chromosomal organization of simple sequence repeats in the Pacific oyster (<Emphasis Type="Italic">Crassostrea gigas</Emphasis>): (GGAT)<Subscript>4</Subscript>, (GT)<Subscript>7</Subscript> and (TA)<Subscript>10</Subscript> chromosome patterns

Chromosome identification is essential in oyster genomic research. Fluorescence in situ hybridization (FISH) offers new opportunities for the identification of oyster chromosomes. It has been used to locate satellite DNAs, telomeres or ribosomal DNA sequences. However, regarding chromosome identification, no study has been conducted with simple sequence repeats (SSRs). FISH was used to probe the physical organization of three particular SSRs, (GGAT)(4), (GT)(7) and (TA)(10) onto metaphase chromosomes of the Pacific oyster, Crassostrea gigas. Hybridization signals were observed in all the SSR probes, but the distribution and intensity of signals varied according to the oligonucleotide repeat. The intercalary, centromeric and telomeric bands were observed along the chromosomes, and for each particular repeat every chromosome pair presented a similar pattern, allowing karyotypic analysis with all the SSRs tested. Our study is the first in mollusks to show the application of SSR in situ hybridization for chromosome identification and karyotyping. This technique can be a useful tool for oyster comparative studies and to understand genome organization in different oyster taxa.     

Karyotyping of comparative genomic hybridization human metaphases using kernel nearest-neighbor algorithm

BACKGROUND: Comparative genomic hybridization (CGH) is a relatively new molecular cytogenetic method that detects chromosomal imbalances. Automatic karyotyping is an important step in CGH analysis because the precise position of the chromosome abnormality must be located and manual karyotyping is tedious and time-consuming. In the past, computer-aided karyotyping was done by using the 4',6-diamidino-2-phenylindole, dihydrochloride (DAPI)-inverse images, which required complex image enhancement procedures. METHODS: An innovative method, kernel nearest-neighbor (K-NN) algorithm, is proposed to accomplish automatic karyotyping. The algorithm is an application of the "kernel approach," which offers an alternative solution to linear learning machines by mapping data into a high dimensional feature space. By implicitly calculating Euclidean or Mahalanobis distance in a high dimensional image feature space, two kinds of K-NN algorithms are obtained. New feature extraction methods concerning multicolor information in CGH images are used for the first time. RESULTS: Experiment results show that the feature extraction method of using multicolor information in CGH images improves greatly the classification success rate. A high success rate of about 91.5% has been achieved, which shows that the K-NN classifier efficiently accomplishes automatic chromosome classification from relatively few samples. CONCLUSIONS: The feature extraction method proposed here and K-NN classifiers offer a promising computerized intelligent system for automatic karyotyping of CGH human chromosomes.     

Chromosomics: Detection of Numerical and Structural Alterations in All 24 Human Chromosomes Simultaneously Using a Novel OctoChrome FISH Assay

Fluorescence in situ hybridization (FISH) is a technique that allows specific DNA sequences to be detected on metaphase or interphase chromosomes in cell nuclei¹. The technique uses DNA probes with unique sequences that hybridize to whole chromosomes or specific chromosomal regions, and serves as a powerful adjunct to classic cytogenetics. For instance, many earlier studies reported the frequent detection of increased chromosome aberrations in leukemia patients related with benzene exposure, benzene-poisoning patients, and healthy workers exposed to benzene, using classic cytogenetic analysis². Using FISH, leukemia-specific chromosomal alterations have been observed to be elevated in apparently healthy workers exposed to benzene^3-6, indicating the critical roles of cytogentic changes in benzene-induced leukemogenesis. Generally, a single FISH assay examines only one or a few whole chromosomes or specific loci per slide, so multiple hybridizations need to be conducted on multiple slides to cover all of the human chromosomes. Spectral karyotyping (SKY) allows visualization of the whole genome simultaneously, but the requirement for special software and equipment limits its application⁷. Here, we describe a novel FISH assay, OctoChrome-FISH, which can be applied for Chromosomics, which we define here as the simultaneous analysis of all 24 human chromosomes on one slide in human studies, such as chromosome-wide aneuploidy study (CWAS)⁸. The basis of the method, marketed by Cytocell as the Chromoprobe Multiprobe System, is an OctoChrome device that is divided into 8 squares, each of which carries three different whole chromosome painting probes (Figure 1). Each of the three probes is directly labeled with a different colored fluorophore, green (FITC), red (Texas Red), and blue (Coumarin). The arrangement of chromosome combinations on the OctoChrome device has been designed to facilitate the identification of the non-random structural chromosome alterations (translocations) found in the most common leukemias and lymphomas, for instance t(9;22), t(15;17), t(8;21), t(14;18)⁹. Moreover, numerical changes (aneuploidy) in chromosomes can be detected concurrently. The corresponding template slide is also divided into 8 squares onto which metaphase spreads are bound (Figure 2), and is positioned over the OctoChrome device. The probes and target DNA are denatured at high-temperature and hybridized in a humid chamber, and then all 24 human chromosomes can be visualized simultaneously. OctoChrome FISH is a promising technique for the clinical diagnosis of leukemia and lymphoma and for detection of aneuploidies in all chromosomes. We have applied this new Chromosomic approach in a CWAS study of benzene-exposed Chinese workers^8,10.