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。     

Cryptic terminal chromosome rearrangements in colorectal carcinoma cell lines detected by subtelomeric FISH analysis

Epithelial tumour karyotypes are often difficult to study by standard cytogenetic methods because of poor chromosome preparation quality and the high complexity of their genomic rearrangements. Subtelomeric fluorescence in situ hybridisation (FISH) has proved to be a useful method for detecting cryptic constitutional chromosomal rearrangements but little is known about its usefulness for tumour cytogenetic analysis. Using a combination of chromosome banding, multicolour karyotyping and subtelomeric FISH, five colorectal cancer cell lines were characterised. The resulting data were compared to results from previous studies by comparative genomic hybridisation and spectral karyotyping or multicolour FISH. Subtelomeric FISH made it possible to resolve several highly complex chromosome rearrangements, many of which had not been detected or were incompletely characterised by the other methods. In particular, previously undetected terminal imbalances were found in the two cell lines not showing microsatellite instability.     

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     

Fluorescence in situ hybridization to chromosomes as a tool to understand human and primate genome evolution

For the last 15 years molecular cytogenetic techniques have been extensively used to study primate evolution. Molecular probes were helpful to distinguish mammalian chromosomes and chromosome segments on the basis of their DNA content rather than solely on morphological features such as banding patterns. Various landmark rearrangements have been identified for most of the nodes in primate phylogeny while chromosome banding still provides helpful reference maps. Fluorescence in situ hybridization (FISH) techniques were used with probes of different complexity including chromosome painting probes, probes derived from chromosome sub-regions and in the size of a single gene. Since more recently, in silico techniques have been applied to trace down evolutionarily derived chromosome rearrangements by searching the human and mouse genome sequence databases. More detailed breakpoint analyses of chromosome rearrangements that occurred during higher primate evolution also gave some insights into the molecular changes in chromosome rearrangements that occurred in evolution. Hardly any "fusion genes" as known from chromosome rearrangements in cancer cells or dramatic "position effects" of genes transferred to new sites in primate genomes have been reported yet. Most breakpoint regions have been identified within gene poor areas rich in repetitive elements and/or low copy repeats (segmental duplications). The progress in various molecular and molecular-cytogenetic approaches including the recently launched chimpanzee genome project suggests that these new tools will have a significant impact on the further understanding of human genome evolution.     

Structural unbalanced chromosome rearrangements resolved by comparative genomic hybridization.

The identification of unbalanced structural chromosome rearrangements using conventional cytogenetic techniques depends on recognition of the unknown material from its banding pattern. Even with optimally banded chromosomes, when large chromosome segments are involved, cytogeneticists may not always be able to determine the origin of extrachromosomal material and supernumerary chromosomes. We report here on the application of comparative genomic hybridization (CGH), a new molecular-cytogenetic assay capable of detecting chromosomal gains and losses, to six clinical samples suspected of harboring unbalanced structural chromosome abnormalities. CGH provided essential information on the nature of the unbalanced aberration investigated in five of the six samples. This approach has proved its ability to resolve complex karyotypes and to provide information when metaphase chromosomes are not available. In cases where metaphase chromosome spreads were available, confirmation of CGH results was easily obtained by fluorescence in situ hybridization (FISH) using specific probes. Thus the combined use of CGH and FISH provided an efficient method for resolving the origin of aberrant chromosomal material unidentified by conventional cytogenetic analysis.     

"Bar-coding" primate chromosomes: molecular cytogenetic screening for the ancestral hominoid karyotype

Two recently introduced multicolor FISH approaches, cross-species color banding (also termed Rx-FISH) and multiplex FISH using painting probes derived from somatic cell hybrids retaining fragments of human chromosomes, were applied in a comparative molecular cytogenetic study of higher primates. We analyzed these "chromosome bar code" patterns to obtain an overview of chromosomal rearrangements that occurred during higher primate evolution. The objective was to reconstruct the ancestral genome organization of hominoids using the macaque as outgroup species. Approximately 160 individual and discernible molecular cytogenetic markers were assigned in these species. Resulting comparative maps allowed us to identify numerous intra-chromosomal rearrangements, to discriminate them from previous contradicting chromosome banding interpretations and to propose an ancestral karyotype for hominoids. From 25 different chromosome forms in an ancestral karyotype for all hominoids of 2N=48 we propose 21. Probes for chromosomes 2p, 4, 9 and Y were not informative in the present experiments. The orangutan karyotype was very similar to the proposed ancestral organization and conserved 19 of the 21 ancestral forms; thus most chromosomes were already present in early hominoid evolution, while African apes and human show various derived changes.     

Cytogenetic characterization of the TM4 mouse Sertoli cell line. I. Conventional banding techniques, FISH and SKY.

Permanent Sertoli cell lines provide an ideal system for the in vitro analysis of function and responsiveness to biochemical/hormonal factors of this particular cell type. In general, cytogenetic analyses of cell lines often reveal remarkable chromosomal changes that may be associated with functional characteristics. In the present study we investigated the mouse Sertoli cell line TM4 by C-banding, silver staining, FISH and spectral karyotyping (SKY). A highly increased chromosome number (average 85-95) as well as five stable marker chromosomes were detected by the conventional staining techniques. SKY identified the markers as a translocation chromosome T(1;3), isochromosomes 11 and 18 and two different-sized microchromosomes. The results show the usefulness of combining SKY and conventional banding methods for the evaluation of chromosome alterations in widely used cell lines.     

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.     

Different chromosome Y abnormalities in Turner syndrome.

A 17-year-old phenotypically female girl was referred for evaluation because of short stature and primary amenorrhea. Cytogenetic analysis showed a mosaic 46,XY/45,X/47,XYY/46,X,idic(Yq)/47,XY,idic(Yq)/48,XXY,idic(Yq)/46,X,t(C;Y) karyotype. Conventional cytogenetic results were supplemented with fluorescence in situ hybridization (FISH) techniques to ensure a better characterization of abnormalities. By using FISH, a supernumerary marker chromosome derived from chromosome Y which could not be detected by conventional cytogenetics was revealed. Furthermore, additional abnormalities and their frequencies were highlighted by the application of DNA probes specific for X and Y chromosomes. Thus, FISH proved useful in determining low frequency cell lines which would need analysis of a large number of good quality metaphase spreads by conventional cytogenetic techniques: it helped in identifying the nature and the origin of unknown markers and rearrangements which have important implication in sexual differentiation and development of gonadal tumours.     

Modern molecular cytogenetic techniques in genetic diagnostics

During the past decade, fluorescence in situ hybridization (FISH) has become an important complementing application in genetic diagnostics. The use of variable FISH techniques enhances the thorough interpretation of numerical and complex chromosome aberrations, bridging the gap between conventional chromosome banding analysis and molecular genetic DNA studies. This review gives a brief overview of the different molecular cytogenetic FISH techniques and applications currently used in routine genetic diagnostics and focus on their advantages and limitations.     

Centromere cleavage is a mechanism underlying isochromosome formation in skin and head and neck carcinomas

Centromeric rearrangements, in the form of isochromosomes or whole-arm translocations, are the most common recurrent changes in head and neck and skin carcinomas. Little is known about the mechanisms behind the origin of these chromosome rearrangements. In the present study, one basal cell carcinoma and two squamous cell carcinomas of the head and neck were thoroughly studied by cytogenetic and fluorescence in situ hybridization techniques. All tumors showed intratumor heterogeneity in the form of cytogenetically related subclones (in all tumors) and unrelated clones (in one tumor). Assessment of karyotypic evolution in these tumors suggests that centromeric cleavage is a mechanism giving rise to isochromosomes. A similar mechanism may also be involved in the formation of whole-arm translocations.     

Primary amenorrhoea in a patient with mosaicism for monosomy X and a derivative X-chromosome

Primary amenorrhoea is defined as the absence of menstruation in phenotypic women aged 16 years or older, if secondary sexual characteristics are present. X chromosome abnormalities probably comprise about one half of all cases, including Turner syndrome and X chromosome rearrangements. Conventional banding cytogenetic methods might miss the accurate detection of structural chromosome abnormalities. The fluorescence in situ hybridization (FISH) and multicolor FISH techniques are required to interpret specific chromosomal rearrangement. As far as we know, we report the first case with chromosome mosaicism for monosomy X and terminal deletion of Xq26 with duplication of Xp11-->pter. In spite of the fact that a 45,X karyotype was detected in 46% of lymphocytes, she was tall and her secondary sexual characteristics were moderately developed, including breast, pubic and axillary hair stages. Cytogenetic and FISH analyses should be considered for patients presenting primary amenorrhoea even if there are no other clinical features suggestive of chromosome abnormality.     

Improvements in cytogenetic slide preparation: controlled chromosome spreading, chemical aging and gradual denaturing

BACKGROUND: Metaphase spreading is an essential technique for clinical and molecular cytogenetics. Results of classical banding techniques as well as complex fluorescent in situ hybridization (FISH) applications, such as comparative genomic hybridization (CGH) or multiplex FISH (M-FISH), are greatly influenced by the quality of chromosome spreading and pretreatment of the slide prior to hybridization. Materials and Methods Using hot steam and a metal plate with a temperature gradient across its surface, a reproducible protocol for slide preparation, aging, and hybridization was developed. RESULTS: This protocol yields good chromosome spreads from even the most difficult cell suspensions and is unaffected by the environmental conditions. Chromosome spreads were suitable for both banding and FISH techniques common to the cytogenetic laboratory. Chemical aging is a rapid slide pretreatment procedure for FISH applications, which allows freshly prepared cytogenetic slides to be used for in situ hybridization within 30 min, thus increasing analytical throughput and reducing benchwork. Furthermore, the gradually denaturing process described allows the use of fresh biologic material with optimal FISH results while protecting chromosomal integrity during denaturing. CONCLUSION: The slide preparation and slide pretreatment protocols can be performed in any laboratory, do not require specialized equipment, and provide robust results.     

Arginase deficiency manifesting delayed clinical sequelae and induction of a kidney arginase isozyme

De novo chromosome structural abnormalities cannot always be diagnosed by the use of standard cytogenetic techniques. We applied a previously developed chromosome-band-specific painting method to the diagnosis of such rearrangements. The diagnostic procedures consisted of microdissection of an aberrant chromosomal region of a given patient, polymerase chain reaction (PCR) amplification of the dissected chromosomal DNA, and subsequent competitive fluorescence in situ hybridization (FISH) using the PCR products as a probe pool on metaphase chromosomes from the patient and/or a karyotypically normal person. With this strategy, we studied 6 de novo rearrangements (6p+, 6q+, 9p+, 17p+, +mar, and +mar) in 6 patients. These rearrangements had been seen by conventional banding but their origin could not be identified. In all 6 patients, we successfully ascertained the origin. Using an aberrant region-specific probe pool, FISH signals appeared on both the aberrant region and a region of another specific chromosome pair. A reverse probe pool that was generated through the microdissection of normal chromosomes at a candidate region for the origin of the aberration hybridized with both the aberrant and the candidate regions. We thus diagnosed one patient with 17p+ as having trisomy for 14q32-qter, one with 9p+ as having trisomy for 12pter-p12, one with 6q+ as having a tandem duplication (trisomy) of a 6q23-q25 segment, one with 6p+ as having a tandem duplication (trisomy) of a 6p23-q21.3 segment, one with a supernumerary metacentric marker chromosome as having tetrasomy for 18pter-cen, and the last with an additional small marker chromosome as having trisomy for 18p11.1 (or p11.2)-q11.2. The present targeted chromosome-band-painting method provides the simple and rapid preparation of a probe pool for region-specific FISH, and is useful for the diagnosis of chromosome abnormalities of unknown origin.     

Multicolor fluorescence in situ hybridization (FISH) applied to FISH-banding

During the last decade not only multicolor fluorescence in situ hybridization (FISH) using whole chromosome paints as probes, but also numerous chromosome banding techniques based on FISH have been developed for the human and for the murine genome. This review focuses on such FISH-banding techniques, which were recently defined as 'any kind of FISH technique, which provide the possibility to characterize simultaneously several chromosomal subregions smaller than a chromosome arm. FISH-banding methods fitting that definition may have quite different characteristics, but share the ability to produce a DNA-specific chromosomal banding'. While the standard chromosome banding techniques like GTG lead to a protein-related black and white banding pattern, FISH-banding techniques are DNA-specific, more colorful and, thus, more informative. For some, even high-resolution FISH-banding techniques the development is complete and they can be used for whole genome hybridizations in one step. Other FISH-banding methods are only available for selected chromosomes and/or are still under development. FISH-banding methods have successfully been applied in research in evolution- and radiation-biology, as well as in studies on the nuclear architecture. Moreover, their suitability for diagnostic purposes has been proven in prenatal, postnatal and tumor cytogenetics, indicating that they are an important tool with the potential to partly replace the conventional banding techniques in the future.     

Multicolor chromosome bar codes

Chromosome bar codes are multicolor banding patterns produced by fluorescence in situ hybridization (FISH) with differentially labeled and pooled sub-regional DNA probes. These molecular cytogenetic tools facilitate chromosome identification and the delineation of both inter- and intra-chromosomal rearrangements. We present an overview of the various conceptual approaches which can be largely divided into two classes: Simple bar codes designed for chromosome identification and complex bar codes for high resolution aberration screening of entire karyotypes. We address the issue of color redundancy and how to overcome this limitation by complementation of bar codes with whole chromosome painting probes.     

A strategy for the characterization of minute chromosome rearrangements using multiple color fluorescence in situ hybridization with chromosome-specific DNA libraries and YAC clones

The identification of marker chromosomes in clinical and tumor cytogenetics by chromosome banding analysis can create problems. In this study, we present a strategy to define minute chromosomal rearrangements by multicolor fluorescence in situ hybridization (FISH) with whole chromosome painting probes derived from chromosome-specific DNA libraries and Alu-polymerase chain reaction (PCR) products of various region-specific yeast artificial chromosome (YAC) clones. To demonstrate the usefulness of this strategy for the characterization of chromosome rearrangements unidentifiable by banding techniques, an 8p+ marker chromosome with two extra bands present in the karyotype of a child with multiple anomalies, malformations, and severe mental retardation was investigated. A series of seven-color FISH experiments with sets of fluorochrome-labeled DNA library probes from flow-sorted chromosomes demonstrated that the additional segment on 8p+ was derived from chromosome 6. For a more detailed characterization of the marker chromosome, three-color FISH experiments with library probes specific to chromosomes 6 and 8 were performed in combination with newly established telomeric and subtelomeric YAC clones from 6q25, 6p23, and 8p23. These experiments demonstrated a trisomy 6pter6p22 and a monosomy 8pter8p23 in the patient. The present limitations for a broad application of this strategy and its possible improvements are discussed.Dedicated to Professor Dr. U. Wolf on the occasion of his 60th birthday     

Integrated cytogenetic map of mitotic metaphase chromosome 9 of maize: resolution,sensitivity, and banding paint development

To study the correlation of the sequence positions on the physical DNA finger print contig (FPC) map and cytogenetic maps of pachytene and somatic maize chromosomes, sequences located along the chromosome 9 FPC map approximately every 10 Mb were selected to place on maize chromosomes using fluorescent in situ hybridization (FISH). The probes were produced as pooled polymerase chain reaction products based on sequences of genetic markers or repeat-free portions of mapped bacterial artificial chromosome (BAC) clones. Fifteen probes were visualized on chromosome 9. The cytological positions of most sequences correspond on the pachytene, somatic, and FPC maps except some probes at the pericentromeric regions. Because of unequal condensation of mitotic metaphase chromosomes, being lower at pericentromeric regions and higher in the arms, probe positions are displaced to the distal ends of both arms. The axial resolution of FISH on somatic chromosome 9 varied from 3.3 to 8.2 Mb, which is 12-30 times lower than on pachytene chromosomes. The probe collection can be used as chromosomal landmarks or as a "banding paint" for the physical mapping of sequences including transgenes and BAC clones and for studying chromosomal rearrangements.     

Fluorescence in situ hybridization (FISH)--application in research and diagnostics

The methods of molecular cytogenetics, in particular fluorescence in situ hybridization (FISH), are widely applied in cytogenetics for identification of numerical and structural chromosomal abnormalities, which are difficult to detect by routine cytogenetic techniques. Due to many advantages, FISH is used in research (gene mapping, gene expression studies, interspecies chromosome homology), and clinical diagnostics (chromosomal aberrations analysis in pre- and postnatal diagnostics, oncology). The techniques of in situ hybridization (ISH) are often employed in addition to classical banding techniques, in case where banding pattern is not reliable. This paper focuses on particular clinical examples, where FISH was successfully used to identify structural and numerical chromosomal aberrations.