Comparative genomic hybridization in clinical cytogenetics.

We report the results of applying comparative genomic hybridization (CGH) in a cytogenetic service laboratory for (1) determination of the origin of extra and missing chromosomal material in intricate cases of unbalanced aberrations and (2) detection of common prenatal numerical chromosome aberrations. A total of 11 fetal samples were analyzed. Seven cases of complex unbalanced aberrations that could not be identified reliably by conventional cytogenetics were successfully resolved by CGH analysis. CGH results were validated by using FISH with chromosome-specific probes. Four cases representing common prenatal numerical aberrations (trisomy 21, 18, and 13 and monosomy X) were also successfully diagnosed by CGH. We conclude that CGH is a powerful adjunct to traditional cytogenetic techniques that makes it possible to solve clinical cases of intricate unbalanced aberrations in a single hybridization. CGH may also be a useful adjunct to screen for euchromatic involvement in marker chromosomes. Further technical development may render CGH applicable for routine aberration screening.     

Characterization of double minute chromosomes’ DNA content in a human high grade astrocytoma cell line by using comparative genomic hybridization and fluorescence in situ hybridization

The presence of double minute chromosomes (dmin) in cancer cells is known to be correlated with gene amplifications. In human high grade astrocytomas or glioblastomas, about 50% of cytogenetically characterized cases display dmin. G5 is a cell line which has been established from a human glioblastoma containing multiple dmin. In order to identify the DNA content of these dmin, three techniques were successively used: conventional cytogenetic analysis, comparative genomic hybridization (CGH), and fluorescent in situ hybridization (FISH). The karyotype of G5 cells showed numerical chromosome changes (hypertriploidy), several marker chromosomes, and multiple dmin. CGH experiments detected two strong DNA amplification areas located in 9p21-22 and 9p24, as well as an underrepresentation of chromosomes 6, 10, 11, 13, 14, and 18q. By using FISH with a chromosome 9-specific painting probe to metaphase chromosomes of the G5 cell line, dmin were shown to contain DNA sequences originating from chromosome 9. This study demonstrates the usefulness of a combination of classical karyotyping, CGH, and FISH to identify the chromosomal origin of amplified DNA sequences in dmin. Received: 30 October 1994 / Revised: 25 February 1996     

Prenatal detection of aneuploidies using fluorescencein situ hybridization: A preliminary experience in an Indian set up

Fluorescencein situ hybridization (FISH) is a powerful molecular cytogenetic technique which allows rapid detection of aneuploidies on interphase cells and metaphase spreads. The aim of the present study was to evaluate FISH as a tool in prenatal diagnosis of aneuploidies in high risk pregnancies in an Indian set up. Prenatal diagnosis was carried out in 88 high-risk pregnancies using FISH and cytogenetic analysis. Multicolour commercially available FISH probes specific for chromosomes 13, 18, 21, X and Y were used. Interphase FISH was done on uncultured cells from chorionic villus and amniotic fluid samples. FISH on metaphase spreads was done from cord blood samples. The results of FISH were in conformity with the results of cytogenetic analysis in all the normal and aneuploid cases except in one case of structural chromosomal abnormality. The hybridization efficiency of the 5 probes used for the detection of aneuploidies was 100%. Using these probes FISH assay yielded discrete differences in the signal profiles between cytogenetically normal and abnormal samples. The overall mean interphase disomic signal patterns of chromosomes 13, 18, 21, X and Y were 94.45%; for interphase trisomic signal pattern of chromosome 21 was 97.3%. Interphase FISH is very useful in urgent high risk cases. The use of FISH overcomes the difficulties of conventional banding on metaphase spreads and reduces the time of reporting. However, with the limited number of probes used, the conventional cytogenetic analysis serves as a gold standard at present. It should be employed as an adjunctive tool to conventional cytogenetics     

Comparative genomic hybridization in combination with flow cytometry improves results of cytogenetic analysis of spontaneous abortions

More than 50% of spontaneous abortions (SAs) have abnormal chromosomes; the most common abnormalities are trisomy, sex chromosome monosomy, and polyploidy. Conventional cytogenetic analysis of SAs depends on tissue culturing and is associated with a significant tissue culture failure rate and contamination by maternally derived cells. Comparative genomic hybridization (CGH), in combination with flow cytometry (FCM), can detect numerical and unbalanced structural chromosomal abnormalities associated with SAs while avoiding the technical problems associated with tissue culture. Routine cytogenetic and CGH analysis was performed independently on tissue from 301 SAs. Samples shown to be chromosomally balanced by CGH were analyzed by FCM to determine ploidy. Of 253 samples successfully analyzed by both approaches, there was an absolute correlation of results in 235 (92.8%). Of the 18 cases with discrepancies between cytogenetic and CGH/FCM results, an explanation could be found in 17. Twelve samples produced a 46,XX karyotype by cytogenetics, whereas CGH/FCM demonstrated aneuploidy/polyploidy or a male genome, indicating maternal contamination of the tissue cultures. In two cases, where tetraploidy was demonstrated by cytogenetics and diploidy by FCM, tissue culture artifact is implied. In three cases, CGH demonstrated an aneuploidy, and cytogenetics demonstrated hypertriploidy. In one unexplainable case, aneuploidy demonstrated by CGH could not be detected by repeat CGH analysis, conventional cytogenetic, or FISH analysis. These results demonstrate that CGH supplemented with FCM can readily identify chromosomal abnormalities associated with SAs and, by avoiding maternal contamination and tissue culture artifacts, can do so with a lower failure rate and more accuracy than conventional cytogenetic analysis.     

Comparative Genomic Hybridization As a New Method for Detection of Genomic Imbalance

Comparative Genomic Hybridization (CGH) is a molecular cytogenetic method for detecting chromosomal imbalances by comparing the copy number of DNA sequences in cells of tested tissue and the reference specimen. CGH is based on two-color fluorescence suppressive in situ hybridization of genomic test and reference DNAs, each labeled with a different fluorochrome, to metaphase chromosomes of a healthy individual. First described by Kallioniemi et al. in 1992, the CGH assay has been widely used for identification and characterization of both numerical and unbalanced structural chromosome abnormalities in cells of different tissues at various pathological conditions in humans, especially in tumor diseases. We discuss the specific features and quality control of comparative genomic hybridization, its advantages and limitations in detection of genomic imbalance and the prospects for development of this technology.     

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     

Genomic imbalances are confined to non-proliferating cells in paediatric patients with acute myeloid leukaemia and a normal or incomplete karyotype

Leukaemia is often associated with genetic alterations such as translocations, amplifications and deletions, and recurrent chromosome abnormalities are used as markers of diagnostic and prognostic relevance. However, a proportion of acute myeloid leukaemia (AML) cases have an apparently normal karyotype despite comprehensive cytogenetic analysis. Based on conventional cytogenetic analysis of banded chromosomes, we selected a series of 23 paediatric patients with acute myeloid leukaemia and performed whole genome array comparative genome hybridization (aCGH) using DNA samples derived from the same patients. Imbalances involving large chromosomal regions or entire chromosomes were detected by aCGH in seven of the patients studied. Results were validated by fluorescence in situ hybridization (FISH) to both interphase nuclei and metaphase chromosomes using appropriate bacterial artificial chromosome (BAC) probes. The majority of these copy number alterations (CNAs) were confirmed by FISH and found to localize to the interphase rather than metaphase nuclei. Furthermore, the proliferative states of the cells analyzed by FISH were tested by immunofluorescence using an antibody against the proliferation marker pKi67. Interestingly, these experiments showed that, in the vast majority of cases, the changes appeared to be confined to interphase nuclei in a non-proliferative status.     

Reverse chromosome painting for the identification of marker chromosomes and complex translocations in leukemia.

BACKGROUND: Chromosome banding techniques and in situ hybridization reveal the majority of chromosomal aberrations. However, difficulties remain in cases of highly contracted chromosomes, poor quality of the metaphases or the presence of markers with the involvement of several chromosomes. Here, it is demonstrated that reverse painting can be applied successfully starting with bone marrow cells from primary acute myelocytic leukemias (AML). METHODS: This was accomplished by culturing the leukemic cells with a cocktail of various growth factors, which yielded sufficient numbers of cells in cycle to harvest chromosomes for sorting. Aberrant chromosomes were flow-sorted and amplified by degenerate oligonucleotide-primed PCR. The resulting products were labeled by nick-translation and hybridized on normal metaphase spreads. RESULTS: Two patients with marker chromosomes in their leukemia cells were analyzed in detail. The hybridization pattern displayed the composition of the aberrant sorted chromosome. Results were compared with conventional cytogenetic analyses that were performed on material obtained from the same aspirate. The reverse-painting technique enabled identification of aberrations that were not detected by conventional cytogenetic analysis. CONCLUSIONS: Primary AML cells can be cultured in vitro, using optimal culture conditions, facilitating the production of high quality flow karyotypes, suitable for sorting of marker chromosomes to produce DOP-PCR derived chromosome painting probes for reverse painting. Valuable additional cytogenetic information can thus be obtained about complex chromosomal rearrangements or structural aberrations that could not be completely resolved by conventional cytogenetic analysis.     

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)].     

Spectral karyotyping analysis of human and mouse chromosomes

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

High-resolution cDNA microarray CGH mapping of genomic imbalances in osteosarcoma using formalin-fixed paraffin-embedded tissue

Formalin-fixed paraffin embedded (FFPE) tumor tissue provides an opportunity to perform retrospective genomic studies of tumors in which chromosomal imbalances are strongly associated with oncogenesis. The application of comparative genomic hybridization (CGH) has led to the rapid accumulation of cytogenetic information on osteosarcoma (OS); however, the limited resolving power of metaphase CGH does not permit precise mapping of imbalances. Array CGH allows quantitative detection and more precise delineation of copy number aberrations in tumors. Unfortunately the high cost and lower density of BACs on available commercial arrays has limited the ability to comprehensively profile copy number changes in tumors such as OS that are recurrently subject to genomic imbalance. In this study a cDNA/EST microarray including 18,980 human cDNAs (which represent all 22 pairs of autosomal chromosomes and chromosome X) was used for CGH analysis of eight OS FFPE. Chromosomes 1, 12, 17, and X harbored the most imbalances. Gain/amplification of X was observed in 4/8 OS, and in keeping with other recent genomic analyses of OS, gain/amplification of 17p11.2 was often accompanied by a distal deletion in the region of the p53 gene. Gain/amplification of the X chromosome was verified using interphase FISH carried out on a subset of OS FFPE sections and OS tissue arrays.     

Comparative genomic hybridization arrays in clinical pathology: progress and challenges

Array-based comparative genomic hybridization (array CGH) genome scanning is a powerful method for the global detection of gains and losses of genetic material in both congenital and neoplastic disorders. When used as a clinical diagnostic test, array CGH combines the whole genome perspective of traditional G-banded cytogenetics with the targeted identification of cryptic chromosomal abnormalities characteristic of fluorescence in situ hybridization (FISH). However, the presence of structural variants in the human genome can complicate analysis of patient samples, and array CGH does not provide morphologic information about chromosome structure, balanced translocations, or the actual chromosomal location of segmental duplications. Identification of such anomalies has significant diagnostic and prognostic implications for the patient. We therefore propose that array CGH should be used as a guide to the presence of genomic structural rearrangements in germline and tumor genomes that can then be further characterized by FISH or G-banding, depending on the clinical scenario. In this article, we share some of our experiences with diagnostic array CGH and discuss recent progress and challenges involved with the integration of array CGH into clinical laboratory medicine.     

Strand-specific fluorescence in situ hybridization: the CO-FISH family

The ability to prepare single-stranded chromosomal target DNA allows innovative uses of FISH technology for studies of chromosome organization. Standard FISH methodologies require functionally single-stranded DNAs in order to facilitate hybridization between the probe and the complementary chromosomal target sequence. This usually involves denaturation of double-stranded probes to induce temporary separation of the DNA strands. Strand-specific FISH (CO-FISH; Chromosome Orientation-FISH) involves selective removal of newly replicated strands from DNA of metaphase chromosomes which results in single-stranded target DNA. When single-stranded probes are then hybridized to such targets, the resulting strand-specific hybridization is capable of revealing a level of information previously unattainable at the cytogenetic level. Mammalian telomeric DNA consists of tandem repeats of the (TTAGGG) sequence, oriented 5'-->3' towards the termini of all vertebrate chromosomes. Based on this conserved structural organization, CO-FISH with a telomere probe reveals the absolute 5'-->3' orientation of DNA sequences with respect to the pter-->qter direction of chromosomes. Development and various applications of CO-FISH will be discussed: detection of cryptic inversions, discrimination between telomeres produced by leading- versus lagging-strand synthesis, and replication timing of mammalian telomeres.     

Atomic force microscopy and scanning near-field optical microscopy studies on the characterization of human metaphase chromosomes

A better knowledge of biochemical and structural properties of human chromosomes is important for cytogenetic investigations and diagnostics. Fluorescence in situ hybridization (FISH) is a commonly used technique for the visualization of chromosomal details. Localizing specific gene probes by FISH combined with conventional fluorescence microscopy has reached its limit. Also, microdissecting DNA from G-banded human metaphase chromosomes by either a glass tip or by laser capture needs further improvement. By both atomic force microscopy (AFM) and scanning near-field optical microscopy (SNOM), local information from G-bands and chromosomal probes can be obtained. The final resolution allows a more precise localization compared to standard techniques, and the extraction of very small amounts of chromosomal DNA by the scanning probe is possible. Besides new strategies towards a better G-band and fluorescent probe detection, this study is focused on the combination of biochemical and nanomanipulation techniques which enable both nanodissection and nanoextraction of chromosomal DNA.     

Subtelomeric rearrangements in Polish subjects with intellectual disability and dysmorphic features

Fluorescent in situ hybridization (FISH) was performed in 76 patients referred to our department because of intellectual disability and dysmorphic features that can be related to subtelomeric microaberrations. In all the patients, conventional cytogenetic methods revealed normal karyotype. Four (5.3%) subtelomeric rearrangements were detected by FISH: 2 subtelomeric 1p36 deletions, an unbalanced translocation involving chromosomes 1 and 12 with 1p36 deletion, and a de novo balanced translocation involving chromosomes 19 and 22. Thus, 3 cases of 1p36 subtelomeric deletion were found (3.95%). To confirm subtelomeric rearrangements in 2 patients, comparative genomic hybridization (CGH) was applied. Moreover, 3 cases of polymorphism without phenotypic effects were found: in 2 patients, the polymorphism involved the long arm of chromosome 2 (maternal derivative in both patients), while in the third patient, a polymorphism of the long arm of chromosome 7 was diagnosed. The latter polymorphism was also found in the patient’s mother and grandfather.     

FISH技术的临床应用研究

荧光原位杂交（FISH）是染色体显带技术的补充和发展,以人类精子,早期胚胎等间期核及中期染色体为材料,用FISH技术检测染色体的数目异常和微小的结构异常。结果快速准确,显示它较之传统的细胞遗传学技术诊断具有明显的优越性,在临床应用中有广泛的前景。     

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     

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。