Improved definition of chromosomal breakpoints using high-resolution multicolour banding

Characterisation of chromosome rearrangements using conventional banding techniques often fails to determine the localisation of breakpoints precisely. In order to improve the definition of chromosomal breakpoints, the high-resolution multicolour banding (MCB) technique was applied to identify human chromosome 5 breakpoints from 40 clinical cases previously assessed by conventional banding techniques. In 30 cases (75%), at least one breakpoint was redefined, indicating that MCB markedly improves chromosomal breakpoint localisation. The MCB pattern is highly reproducible and, in contrast to conventional banding pattern, is consistent in both short and elongated chromosomes. This might be of fundamental interest for the detection of chromosomal abnormalities, especially in tumour cells. Moreover, MCB even allows the detection of abnormalities that remain cryptic in GTG-banding analysis.     

mBAND analysis for high- and low-LET radiation-induced chromosome aberrations: a review

During long-term space travel or cancer therapy, humans are exposed to high linear energy transfer (LET) energetic heavy ions. High-LET radiation is much more effective than low-LET radiation in causing various biological effects, including cell inactivation, genetic mutations, cataracts and cancer induction. Most of these biological endpoints are closely related to chromosomal damage, and cytogenetic damage can be utilized as a biomarker for radiation insults. Epidemiological data, mainly from survivors of the atomic bomb detonations in Japan, have enabled risk estimation from low-LET radiation exposures. The identification of a cytogenetic signature that distinguishes high- from low-LET exposure remains a long-term goal in radiobiology. Recently developed fluorescence in situ hybridization (FISH)-painting methodologies have revealed unique endpoints related to radiation quality. Heavy-ions induce a high fraction of complex-type exchanges, and possibly unique chromosome rearrangements. This review will concentrate on recent data obtained with multicolor banding in situ hybridization (mBAND) methods in mammalian cells exposed to low- and high-LET radiations. Chromosome analysis with mBAND technique allows detection of both inter- and intrachromosomal exchanges, and also distribution of the breakpoints of aberrations.     

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。     

Reorganization of the X chromosome in voles of the genus Microtus

Comparative chromosomal analysis is a powerful tool in the investigation of the mechanisms of chromosomal evolution. The accuracy of the analysis depends on the availability of region-specific markers to follow the fate of the particular chromosomal region through the evolution of species. We have assigned 12 unique sequences to the euchromatic part of the vole X chromosome, which serve as reliable markers of chromosomal segments. Together with region-specific libraries and GTG banding, these markers allow us to delineate the homologous regions of the X chromosomes in five species of the genus Microtus. We found that X chromosomes of these species differ by numerous rearrangements and all rearrangements are clustered at specific breakpoints. Moreover, these breakpoints were found to colocalise with repetitive and/or duplicated DNA sequences. We suggest that clusters of repeated and/or duplicated DNA sequences have played a crucial role in the formation of rearrangement hot spots during evolution of the X chromosome in the subgenus Microtus.     

Chromosomal banding patterns and karyotype evolution in three pig kidney cell strains (PK15, F and RP)

Some of the techniques used to obtain banding patterns in human karyotype are adapted here to three pig kidney cell strains (PK15, F and RP). These strains were established respectively in 1955, 1962 and 1969. The banding techniques used are: controlled heating, ASG technique, alkaline treatment and proteolytic digestion with trypsin or pronase. Knowing the specific banding of the pig karyotype, it has been possible to study the chromosomal rearrangements observed in the heteroploid cell strains. If the strain is old, the rearrangements are more numerous. However, they are the same as the ones usually described: in the three strains, one of the two chromosomes of each pair is retained unchanged as judged by its banding. The other chromosome is either present, lost or modified. It may constitute part of a marker chromosome.     

In situ hybridization and translocation breakpoint mapping. II. Two unusual t(21;22) translocations

Using a combination of banding techniques, we examined two atypical 21;22 translocations, 46,XX or XY,t(21;22)(p11;q11). In situ chromosomal hybridization of a probe for the constant region of the lambda light chain locus demonstrated that the 22q11 breakpoints of both rearrangements were proximal to the C lambda gene cluster. These studies permitted us to distinguish the 22q11 breakpoints of these translocations from the breakpoint of the 22q--chromosome of chronic myelogenous leukemia.     

mBAND analysis of chromosomal aberrations in human epithelial cells exposed to low- and high-LET radiation

Energetic heavy ions pose a potential health risk to astronauts who have participated in extended space missions. High-LET radiation is much more effective than low-LET radiation in the induction of biological effects, including cell inactivation, genetic mutations, cataracts and cancer. Most of these biological end points are closely correlated with chromosomal damage, which can be used as a biomarker for radiation damage. Multicolor banding in situ hybridization (mBAND) has proven to be highly useful for the study of intrachromosomal aberrations, which have been suggested as a biomarker of exposure to high-LET radiation. To investigate biological signatures of radiation quality and the complexity of intrachromosomal aberrations, we exposed human epithelial cells in vitro to (137)Cs gamma rays or iron ions (600 MeV/nucleon) and collected chromosomes using a premature chromosome condensation technique. Aberrations in chromosome 3 were analyzed using mBAND probes. The results of our study confirmed the observation of a higher incidence of inversions for high-LET radiation. However, detailed analysis of the inversion type revealed that both iron ions and gamma rays induced a low incidence of simple inversions. Half of the inversions observed in the low-LET-irradiated samples were accompanied by other types of intrachromosome aberrations, but few inversions were accompanied by interchromosome aberrations. In contrast, iron ions induced a significant fraction of inversions that involved complex rearrangements of both inter- and intrachromosome exchanges.     

Correlating breakage-fusion-bridge events with the overall chromosomal instability and in vitro karyotype evolution in prostate cancer

Chromosomal instability (CIN) is thought to underlie the generation of chromosomal changes and genomic heterogeneity during prostatic tumorigenesis. The breakage-fusion-bridge (BFB) cycle is one of the CIN mechanisms responsible for characteristic mitotic abnormalities and the occurrence of specific classes of genomic rearrangements. However, there is little detailed information concerning the role of BFB and CIN in generating genomic diversity in prostate cancer. In this study we have used molecular cytogenetic methods and array comparative genomic hybridization analysis (aCGH) of DU145, PC3, LNCaP, 1532T and 1542T to investigate the in vitro role of BFB as a CIN mechanism in karyotype evolution. Analysis of mitotic structures in all five prostate cancer cell lines showed increased frequency of anaphase bridges and nuclear strings. Structurally rearranged dicentric chromosomes were observed in all of the investigated cell lines, and Spectral Karyotyping (SKY) analysis was used to identify the participating rearranged chromosomes. Multicolor banding (mBAND) and aCGH analysis of some of the more complex chromosomal rearrangements and associated amplicons identified inverted duplications, most frequently involving chromosome 8. Chromosomal breakpoint analysis showed there was a higher frequency of rearrangement at centromeric and pericentromeric genomic regions. The distribution of inverted duplications and ladder-like amplifications was mapped by mBAND and by aCGH. Adjacent spacing of focal amplifications and microdeletions were observed, and focal amplification of centromeric and end sequences was present, particularly in the most unstable line DU145. SKY analysis of this line identified chromosome segments fusing with multiple recipient chromosomes (jumping translocations) identifying potential dicentric sources. Telomere free end analysis indicated loss of DNA sequence. Moreover, the cell lines with the shortest telomeres had the most complex karyotypes, suggesting that despite the expression of telomerase, the reduced telomere length could be driving the observed BFB events and elevated levels of CIN in these lines.     

Rapid Identification of Chromosomal Abnormalities in Human Neoplasia

Recent advances in banding techniques have led to the belief that certain chromosomal defects are consistently associated with specific types of human neoplasia. Based on the GTG technique, it has been suggested that the malignant cells of most neoplasias show chromosomal abnormalities (Yunis et al. 1983). From this recent publication of Yunis it appears that the majority of bands involved in carcinogenesis are G-negative, i.e., do not stain by the GTG technique, and it is therefore difficult to localize the breakpoints. In some of our recent publications we emphasized the importance of the RFA technique (Verma and Lubs 1975), which stains Giemsa-negative bands darkly, thus providing precise identification of chromosomal abnormalities (Verma and Dosik 1976). However, this technique cannot be applied until the slides have aged for at least 7 days. Therefore, we are reporting an alternative procedure using BrdU which provides “reverse” banding immediately when the slides are stained with acridness orange and examined with a fluorescence microscope.     

Array painting using microdissected chromosomes to map chromosomal breakpoints

Molecular characterization of breakpoints of chromosomal rearrangements is a successful strategy for the identification of candidate disease genes. Mapping translocation breakpoints and rearranged chromosomal boundaries is labor intensive and/or time consuming. Here, we present a novel and rapid procedure to map such chromosomal breakpoints by hybridizing amplified microdissection derived DNA of aberrant chromosomes to arrays containing genomic clones. We illustrate the potential of the technique by molecularly delineating the breakpoints in five small supernumerary marker chromosomes (sSMC) and mapping the breakpoints of five different chromosomal translocations.     

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.     

Chromosome mapping of Miller-Diecker,Smith-Magenis and RARA loci in non-human primates: implications in the evolution of human chromosome 17

Molecular cytogenetics allows to verify chromosomal homologies previously hypothesised on the base of banding pattern comparison in different species. So far only the chromosome painting technique has been extensively used in studies of chromosomal evolution. This technique allows to detect only interchromosomal rearrangements. Human and Great Apes chromosomes basically differ by intrachromosomal rearrangements, in particular inversions; with chromosome painting it has just been possible to confirm the origin by fusion of human chromosome 2 and a reciprocal translocation in Gorilla, involving the homologous of chromosome 5 and 17. In order to verify intrachromosomal rearrangements in human chromosomal evolution, chromosome mapping of human loci in non-human primates is a useful approach. We mapped Miller-Diecker, Smith-Magenis and RARA loci localised on human chromosome 17, in Gorilla gorilla, Pongo pygmaeus, Macaca fascicularis and Cercopithecus aethiops. On the base of the obtained results it was possible to verify chromosomal rearrangements previously identified by banding, to achieve new informations about the controversial evolution of human chromosome 17, and to detect the occurrence of a paracentric inversion in the homologous in Cercopithecus aethiops.     

Karyotype analysis of 2 species of gibbons (Hylobates lar and H. concolor) with different banding species]

The mitotic chromosomes of two species of gibbons (Hylobates lar and H. concolor) are examined and compared, using various banding techniques. These two species have very different karyotypes. At the most, seven pairs of chromosomes have a similar banding pattern. The other elements generally differ by complex structrual rearrangements. Thus, it is difficult to propose a scheme for chromosomal evolution at this stage. Comparison with the karyotypes of man and anthropoid apes also shows very important differences; very few chromosomes are common or only slightly modified. Some considerations about the hypothetical origin of particular chromosomal structures are given.     

Integrated cytogenetic and high-resolution array CGH analysis of genomic alterations associated with MYCN amplification

Amplification of oncogenes and closely linked flanking genes is common in some types of cancer and can be associated with complex chromosome rearrangements and/or co-amplification of non-syntenic chromosomal regions. To better understand the etiology and structural complexity of focal MYCN amplicons in human neuronal cancer, we investigated the precise chromosomal locations of high copy number genomic regions in MYCN amplified cell lines. An integrated cytogenetic map of the MYCN amplicon was created using high-resolution array CGH, spectral karyotyping (SKY), multi-color banding (mBAND), and fluorescence in situ hybridization (FISH) in 4 human neuronal tumor cell lines. The evidence of complex intra- and inter-chromosomal events, providing clues concerning the nature of the genomic mechanisms that contributed to the process of MYCN amplification, was observed. The presence of multiple co-amplified syntenic or non-syntenic sequences in the MYCN amplicon is quite intriguing. MYCN is usually centrally located in the amplicon; however, the structure and complexity of the amplicons were highly variable. It is noteworthy that clusters of unstable repetitive regions characterized by CNV sequences were present throughout the regions encompassed by MYCN gene amplification, and these sequences could provide a mechanism to destabilize this region of the genome. Complex structural rearrangements involving genomic losses and gains in the 2p24 region lead to MYCN amplification and that these rearrangements can trigger amplification events.     

Trypsin G- and C-banding for interchange analysis and sex identification in the chicken

The trypsin banding methods were applied to feather pulp and embryonic material of the chicken. Two contrasting types of chromosomal banding patterns were obtained by varying the duration of trypsin treatment. A short time treatment shows a G-banding pattern which has characteristic and distinctive bands along the chromosome arms. Prolonging the trypsin treatment causes the G-banding pattern to disappear, and only the centromeres and the W chromosome remained heterochromatic which is characteristic of the C-banding pattern. The application of the G-banding pattern analysis was used to identify regions of chromosomes involved in rearrangements. The simplified trypsin technique which produces the C-banding pattern makes it relatively easy to identify the W sex-chromosome and determine sex in avian species.     

A complex chromosomal rearrangement detected prenatally and studied by fluorescence in situ hybridization

We report of case of a complex chromosomal rearrangement detected prenatally and studied with traditional banding methods and fluorescence in situ hybridization. The combination of these techniques showed that four chromosomes were involved in the translocation. Nine breakpoints were proposed to explain these results. Some of the findings could only be detected with fluorescence in situ hybridization, demonstrating the usefulness of this technique in characterizing chromosomal abnormalities that would otherwise be difficult to interpret correctly with classical cytogenetics alone.     

FISH diagnostics

For over two decades banding has remained the "gold standard" of cytogenetic analysis, providing the first genome-wide screen for abnormalities. However, conventional cytogenetic banding techniques are limited to the detection of rearrangements involving more than 2 Mb of DNA. In addition,the identification of de novo unbalanced chromosome rearrangements provides a particular challenge for chromosome banding to decipher. In recent years a number of techniques based on FISH have evolved, all of which complement the conventional banding approaches and which have steadily increased the accuracy of cytogenetic diagnosis. FISH is now the method of choice because of the increased sensitivity, and speed with which it can be applied to a variety of cellular targets. In this article we try to highlight the technical aspects of FISH and the practical application of this technique on different tumors (soft tissue tumors, breast carcinomas, renal cell carcinomas, bladder tumors and germ cell tumors).     

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.     

Diagrammatic representation for chromosomal mutagenesis studies IV. Radiation-induced rearrangements in Ateles sp. (Primate,Platyrhini)

In order to study the induction of rearrangements by γ-rays in relation to chromosomal size and morphology, experiments were conducted in an Ateles, a species with a rather unusual karyotype among primates. It possesses some very large chromosomes, which tend to be too rarely affected, especially by intrachanges like inversions. Both their large size and their characteristic banding pattern suggest that this low involvement is not due to difficulty of analysis. This suggests very strongly that chromosomal involvement in rearrangements is not a function of size. The possible role of other factors involved in chromosomal rearrangements like chromosome position during interphase are discussed.