Novel molecular cytogenetic ... molecular... · Molecular cytogenetic techniques that are based on...

Accession information: (00)00194-0a.pdf (short code: txt001trn); 14 September 2000ISSN 1462-3994 Published by Cambridge University Press

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expert reviewsin molecular medicine

Novel molecular cytogenetic techniques

for identifying complex chromosomal

rearrangements: technology and

applications in molecular medicine

Nicole McNeil and Thomas Ried

Nicole McNeil, Biologist, Department of Genetics, Medicine Branch, Division of Clinical Sciences,National Cancer Institute, National Institutes of Health, Building 9, Room 1N105, 9 Memorial Drive,Bethesda, MD 20892, USA. Tel: +1 301 402 2008; Fax: +1 301 402 1204; E-mail: [email protected]

Thomas Ried, Senior Investigator, Department of Genetics, Medicine Branch, Division of ClinicalSciences, National Cancer Institute, National Institutes of Health, Building 9, Room 1N105,9 Memorial Drive, Bethesda, MD 20892, USA. Tel: +1 301 402 2008; Fax: +1 301 402 1204;E-mail: [email protected]

Laboratory website: http://www.riedlab.nci.nih.gov

Molecular cytogenetic techniques that are based on fluorescence in situhybridisation (FISH) have become invaluable tools for the diagnosis andidentification of the numerous chromosomal aberrations that are associatedwith neoplastic disease, including both haematological malignancies andsolid tumours. FISH can be used to identify chromosomal rearrangements,by detecting specific DNA sequences with fluorescently labelled DNA probes.The technique of comparative genomic hybridisation (CGH) involves two-colourFISH. It can be used to establish ratios of fluorescence intensity values betweentumour DNA and control DNA along normal reference metaphase chromosomes,and thereby to detect DNA copy-number changes such as gains and losses ofspecific chromosomal regions and gene amplifications. Spectral karyotyping(SKY) is a novel molecular cytogenetic method for characterising numericaland structural chromosomal aberrations. SKY involves the simultaneoushybridisation of 24 differentially labelled chromosome-painting probes, followedby spectral imaging and chromosome classification, and produces a colourkaryotype of the entire genome. The use of SKY has contributed significantlyto the identification of chromosomal anomalies that are associated withconstitutional and cancer cytogenetics, and has revealed many aberrationsthat go undetected by traditional banding techniques. In this article, we havereviewed these new molecular cytogenetic techniques and described theirvarious applications in molecular medicine.



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The study of human chromosomes has beengoing on for well over a 100 years. However,progress has been impeded by difficultiesassociated with the preparation of high-qualitychromosomes from mammalian cells. It was notuntil 1956 that Tjio and Levan reported thecorrect number of human chromosomes (Ref. 1).In the 1960s, the addition of phytohaemagglutininto blood lymphocyte cultures was found tostimulate cell division (i.e. mitosis), significantlyincreasing the number of metaphase spreads(Refs 2, 3). These discoveries improved theanalysis of human chromosomes and led to therealisation that the analysis of chromosomes wasindispensable to the study of human disease. Afterthe observation of an extra copy of chromosome21 (i.e. trisomy) in patients suffering fromDown syndrome, other trisomies were found inthe D and G group chromosomes and were linkedto certain characteristic phenotypes that areassociated with Patau syndrome and Edwardssyndrome, respectively. Another milestone wasthe discovery of the Philadelphia chromosomeand its association with chronic myelogenousleukaemia (Ref. 4), which invigorated the field ofcancer cytogenetics.

The 1970s saw the introduction and successfulapplication of a variety of staining techniquesthat gave chromosomes a banding pattern.Banding, by greatly improving the accuracy ofchromosome analysis (Refs 5, 6), permitted theanalysis of many different tissues and diseases.Such banding patterns facilitated the analysisof subchromosomal regions. High-resolutionbanding methodology made possible the detectionof chromosomal deletions and the identificationof small translocations (Ref. 7).

Until the advent of molecular cytogenetictechniques in the 1980s, chromosomal analysishad relied on the study of chromosomalbands. However, it was often difficult todiscern the origins of the chromosomes that wereinvolved in chromosomal rearrangements (e.g.translocations), particularly in the case of solidtumours and leukaemia. Modern cytogenetictechniques have improved the diagnosis ofchromosomal aberrations. Fluorescence in situhybridisation (FISH) probes have been designedto detect specific regions of DNA, and thus toelucidate abnormalities even at the level of thegene, which cannot be detected by conventionalbanding techniques. Some of the benefits ofFISH include the confirmation of chromosome

breakpoints, an assessment of specific nucleic acidsequences and the ability to detect such sequencesin non-dividing cells (i.e. interphase cytogenetics).Although FISH is an extremely useful technique,until recently only a few target sequences couldbe visualised simultaneously.

By contrast, comparative genomic hybridisation(CGH) is a molecular cytogenetic techniquethat screens for whole-genomic imbalances intumour samples. It identifies chromosomalgains and losses (e.g. deletions, duplications oramplifications) by using differentially labelledtumour DNA and normal DNA (Ref. 8). Onvisualising the two different fluorochromes (i.e.molecules that are fluorescent when appropriatelyexcited), differences in the intensity of fluorescencealong the chromosome correspond to the loss orgain of genetic material in the tumour sample.

Spectral karyotyping (SKY) is a technique thatcombines the power of conventional chromosomeanalysis with the specificity of FISH. SKY canbe used to identify marker chromosomes (i.e.chromosomes that are important in the diagnosisof a disease) and detect telomeric translocations,which are sometimes difficult to identify usingtraditional banding analysis. This technique hasalso proven to be beneficial in elucidating thecomplex rearrangements that are observed incancer genomes (for examples, see Refs 9, 10,11, 12). SKY entails a single multicolour FISHanalysis, which can be used to yield 24different-coloured chromosomes in a humanmetaphase spread. SKY involves a combinationof epifluorescence microscopy, charge-coupleddevice (CCD) imaging and Fourier spectroscopyto measure the complete emission spectra at allimage points (Refs 13, 14).

These three molecular cytogenetic techniquesand their applications in the analysis ofchromosomal aberrations that are associatedwith human disease are discussed in moredetail below.

Fluorescence in situ hybridisation(FISH)

The FISH technique is based on the discovery thatlabelled ribosomal RNA hybridises to acrocentricchromosomes (i.e. chromosomes that have anon-centrally located centromere, and thereforeunequal chromosome arms; Refs 15, 16). Initially,radioactive isotopes were used for this technique;however, the use of fluorochromes is safer,requires a shorter reaction time and can give rise



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to different colours. FISH involves a fluorescentlylabelled DNA probe being hybridised to genomicDNA sequences, and can be used to study aspecific site on a chromosome.

Methodology of FISHThe first consideration in FISH is the availabilityof a DNA probe. Once pure DNA has been isolated,a fluorochrome must be incorporated into theDNA probe in a reaction known as labelling. DNAprobes can be labelled by enzymatic proceduressuch as nick translation, random priming or thepolymerase chain reaction (PCR). During nicktranslation, the exonuclease activity of DNApolymerase causes a single-strand break (ornick) in the DNA, by removing a nucleotide(i.e. adenine, guanine, thymidine or cytosine).Subsequently, a specific fluorescently labellednucleotide is incorporated into the nickedstrand by DNA polymerase, which uses the DNAsequence of the non-nicked strand as a template.When a probe is produced from genomic DNA,Cot-1 DNA is added to the mixture. HumanCot-1 DNA consists of the repetitive sequencesof the genome, and it is used to suppress thehybridisation of repetitive sequences. Failure touse Cot-1 DNA often results in non-specifichybridisation, making it difficult to distinguishthe ‘signal’ from the ‘background noise’.

Metaphase chromosome spreads are preparedby using a spindle inhibitor such as Colcemid toarrest cultured cells during mitosis. Hypotonicsolution (0.075 M potassium chloride) andfixative (methanol and acetic acid in a 3:1ratio) are then applied sequentially. Finally,the chromosome suspension is dropped ontoglass slides (Ref. 17). Before hybridisation,metaphase chromosomes and interphase nucleiare pretreated enzymatically to enhance theiraccessibility to the probe and to reduce the amountof cytoplasm (Ref. 18). The pretreated slide isheated to denature the DNA.

The previously prepared probe and Cot-1DNA are mixed and denatured together andthen applied to the slide for hybridisation.Denaturation of the probe and Cot-1 DNA allowsthe repetitive sequences to anneal to the Cot-1DNA before hybridisation. This can then befollowed by the site-specific binding of the single-copy probe sequences to the denatured targetDNA. Hybridisation between the probe andtarget DNA (on the slide) takes place during anincubation period of ~16–48 hours at 37°C. This

incubation time can vary depending on the probesused: shorter hybridisation times can be used forrepetitive DNA probes or chromosome-paintingprobes, whereas longer incubation times areneeded for the hybridisation of complementaryDNA (cDNA) sequences or complete genomes.

Detecting the probe permits the visualisationof target DNA sequences. Detection startswith post-hybridisation washes of the slide informamide and SSC (sodium chloride, sodiumcitrate) salt solutions to remove any excessprobe that is non-specifically bound. However,a detection step is not required for all DNAprobes. In a direct-label reaction, a fluorochrome-conjugated nucleotide [e.g. 2'-deoxyuridine 5'triphosphate (dUTP) conjugated to fluoresceinisothiocyanate (FITC)] is incorporated into thestrands. In an indirect-label reaction, reportermolecules such as biotin or digoxigenin areincorporated into the DNA. Such indirectlabels require a detection step in which thereporter molecule or hapten is labelled withan agent such as avidin or anti-digoxin thatis conjugated to a fluorochrome. The detectionmethods for biotinylated probes employavidin–fluorochrome conjugates, whereas thosefor digoxigenin-labelled probes employ anti-digoxin–fluorochrome conjugates.

FISH microscopyFISH signals are visualised by fluorescencemicroscopy using a light source that illuminatesthe fluorescently labelled specimens. Mercuryvapour lamps and xenon lamps both emit lightthat excites fluorochromes. To visualise thefluorescence signal of the probe, a variety ofspecific filter sets can be used to separate thedifferent fluorochromes (Refs 19, 20, 21). Digitalimaging systems, such as a CCD camera, capturethe image and quantify fluorescent signals(Ref. 22). Resultant images are analysed oncommercially available systems (e.g. from Vysis,Leica Microsystems or Applied Spectral Imaging;see box entitled ‘Further reading, resources andcontacts’).

Clinical applications of FISHConventional high-resolution chromosomebanding techniques as used in cytogeneticlaboratories can result in up to 1000 bands pergenome; however, even at such high resolution,disease-associated deletions, duplications ortranslocations can be difficult to discern. By



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contrast, FISH can be used to determine the originof marker chromosomes and to confirm numericaland structural aberrations. Using bandingtechniques on highly extended chromosomes, thesmallest chromosome abnormality detectable is~2000–3000 kilobases (kb), whereas FISH probescan detect regions as small as 0.5 kb on metaphasechromosomes. The increased resolution providedby FISH has particular relevance in the studyof microdeletion syndromes (e.g. Prader-Willi)because the size of the DNA region that has beendeleted is often too small to be detected byconventional banding techniques.

FISH probes are highly specific for their targetor cDNA sequence, and can be divided into fourmain types: gene-specific probes, repetitive-sequence probes, whole-genomic DNA probesand chromosome-painting probes (see Fig. 1).

Gene-specific probesGene-specific probes target specific nucleic acidsequences within chromosomes. Examples of suchprobes include bacterial artificial chromosome(BAC) and yeast artificial chromosome (YAC)probes and cosmids. These probes have proven

particularly useful in the study of microdeletionsyndromes, where the absence of a gene often goesundetected by conventional banding methods.For instance, Duchenne muscular dystrophy is aprogressive muscular degenerative disease thatresults from a deletion at Xp21 (i.e. band 21on the short arm of the X chromosome) inaffected males. Although the deletion can varyin size, exon 45 of the dystrophin gene ismissing in 60% of patients. FISH probes thatcan detect deleted regions of this gene areelegant tools for determining the carrier status ofclinically unaffected women (Refs 23, 24, 25).Gene-specific probes are also useful for mappinggenes on chromosomes.

Repetitive-sequence probesRepetitive-sequence probes bind to regions thatare rich in repetitive base-pair sequences.Examples of such probes include centromeric andtelomeric probes. Centromeres frequently containA–T-rich tandem repeats, whereas telomeres arerecognised by the short repetitive sequenceTTAGGG. Centromeric probes have applicationsin the identification of marker chromosomes

Figure 1. Examples of different types of fluorescence in situ hybridisation (FISH) probes. (a) Gene-specific probes target specific nucleic acid sequences on a chromosome. (b) Centromeric probes bind torepetitive sequences that are specific to the centromeric regions. (c) Telomeric probes recognise the repetitivesequence TTAGGG, and can be used to visualise all telomeres simultaneously. Chromosome-specific telomericprobes hybridise to subtelomeric, chromosome-specific repeats. (d) Chromosome-painting probes consist ofpools of chromosome-specific probes (fig001trn).

Gene-specificprobe

Centromericprobe

Repetitive-sequence probes

Telomericprobe

Chromosome-paintingprobe

a b c d

Expert Reviews in Molecular Medicine © 2000 Cambridge University Press

Examples of different types of fluorescence in situ hybridisation (FISH) probes



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and numerical chromosome abnormalities ininterphase nuclei and when specimens are sexmismatched. Telomeric probes and subtelomere-specific probes (Ref. 26) are commonly used toidentify cryptic chromosomal translocations suchas those occurring in cases of unknown mentalretardation.

Whole-genomic DNA probesWhole-genomic DNA probes are used for theFISH-based technique CGH (see below). They canbe used to detect genomic imbalances in tumourgenomes by combining tumour and normal DNAto analyse gains and losses.

Chromosome-painting probesChromosome-painting probes contain sequencesthat are specific to either a single chromosome(i.e. whole-chromosome-painting probes) oran arm of a chromosome (i.e. chromosome-arm-painting probes). After hybridisation, oneor more chromosomes of interest are ‘lit up’in different colours, which are dependent onwhich particular fluorochromes have been used(see Fig. 2). This technique is particularly usefulfor identifying chromosome arms that areinvolved in translocations, as well as for markerchromosomes and ring chromosomes. Ringchromosomes are formed when breaks occur inthe short and long arms of a chromosome, andthe broken ends re-join to form a ring.

Whole-chromosome-painting probes are alsoused in SKY (as discussed later). By assigningan individual colour to each of the 23 pairs ofhuman chromosomes, an entire karyotype (i.e.all 46 chromosomes) can be differentiallylabelled. As well as being useful for visualisingall aberrations in a simple experiment, SKY canbe used to elucidate cryptic translocations thatoften go undetected by conventional cytogeneticanalysis.

Comparative genomic hybridisation(CGH)

CGH involves the differential labelling oftest and reference DNA to measure geneticimbalances in entire genomes. CGH is based onquantitative two-colour FISH (Ref. 27). It hasbecome an invaluable technique for studyingchromosomal aberrations that occur in solidtumours and other malignancies. A majoradvantage of the CGH technique is that only DNAfrom the tumour samples is needed for analysis;

this avoids the often-difficult preparation oftumour metaphase chromosomes, which canhave a poor morphology and resolution.Instead, karyotypically normal metaphasechromosomes are used to detect tumour-associated chromosomal gains and losses.Another advantage of CGH is that formalin-fixedtissue sections can be used; thus, comparisons canbe made between a phenotype and genotype, andgenetic changes can be correlated with the clinicalcourse of a disease.

Figure 2. Fluorescence in situ hybridisation(FISH) using whole-chromosome-paintingprobes. Chromosome-painting probes were usedto rule out the presence of an extra copy ofchromosome 19 (i.e. trisomy 19) in this metaphasespread, which was derived from a glial cell takenfrom the brain of a mouse. Chromosome 19 appearsgreen [it was labelled with avidin conjugated tofluorescein isothiocyanate (FITC)] against the bluebackground stain of 4,6-diamidino-2-phenylindole(DAPI). Chromosome 13, which was used as anormal control chromosome, has also beenidentified and appears red [it was labelled with avidinconjugated to tetramethylrhodamine isothiocyanate(TRITC)] (fig002trn).

Expert Reviews in Molecular Medicine 2000 Cambridge University Press

Fluorescence in situ hybridisation (FISH)using whole-chromosome-painting probes

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Methodology of CGHFor CGH, whole-genomic DNA is isolated froma tumour by standard extraction protocols.Control or reference DNA is isolated from anindividual who has either a normal 46, XXkaryotype or a normal 46, XY karyotype. TheDNA that has been extracted from the twogenomes is differentially labelled (for exampleusing biotin conjugated to dUTP for the tumourgenome and digoxigenin conjugated to dUTPfor the normal genome). The tumour andnormal DNA samples are combined, and anexcess of unlabelled human Cot-1 DNA is addedinto the hybridisation mixture, to suppress therepetitive sequences that are present in bothgenomes. The Cot-1 DNA is essential becausehybridisation of the repetitive DNA wouldimpair the evaluation of the unique sequences thatare either overrepresented or underrepresentedin the tumour genome. This probe mixture ishybridised to normal human reference metaphasechromosomes. Avidin coupled with FITC (whichfluoresces green) is used for the detection ofbiotin-labelled tumour DNA, whereas anti-digoxin coupled to rhodamine (which fluorescesred) is used for the digoxigenin-labelledcontrol DNA. The relative colour intensities ofthe two flurochromes reflect DNA copy-numberalterations in the tumour genome (Ref. 8; seeFig. 3).

CGH analysisTo determine copy-number imbalances withina tumour, CCD images must be acquired fromseveral metaphase spreads. Variations in thefluorochrome intensity along the chromosomes ina metaphase spread reflect copy-number changes(i.e. gains and losses of DNA along a chromosome)in the tumour sample. For example, using thefluorochromes mentioned above, a loss of DNAwithin the tumour genome shifts the colour ofthat region to red. A gain of a chromosomal regionwould be shown by an increased intensity of thegreen fluorescence in the reference metaphasepreparation. If chromosomes or subchromosomalregions are balanced with respect to DNA contentin both tumour and control samples, the intensityof the red and green fluorescence is similar.Digital image analysis must be used to quantifyfluorochrome intensity, especially in cases wherechanges involve low copy numbers or wheremany gains and losses have occurred. A CCDcamera and fluorochrome-specific optical filters

are used for image acquisition with rhodamineand FITC labels. A variety of filter sets can bepurchased through commercial vendors (e.g.Chroma Technology Corp.). Specialised softwareselects metaphases that have an adequate signalintensity, segments them along the chromosomalaxis, and correctly identifies and orientates eachchromosome. For each chromosome, a ratio valueof fluorochrome intensity is generated from aminimum of five metaphase spreads. Finally, anaverage ratio profile for each chromosome isproduced, indicating the calculated gains andlosses (Refs 28, 29; see Fig. 4).

Experimental investigations with CGHCGH is useful for the analysis of chromosomalgains and losses in solid tumours. Recurrentpatterns of gains and losses have been observedin the malignant tissue from virtually all humancancers (Refs 30, 31, 32, 33). For example, usingCGH to study pancreatic carcinomas, Ghadimiand colleagues found recurrent gains on thechromosome arms 3q, 5p, 7p, 8q, 12p and 20q.Losses were observed on the chromosome arms8p, 9p, 17p, 18q and 19p and on chromosome 21(Ref. 10). Heselmeyer and colleagues studiedtissue from the cervical epithelium at variousstages of dysplasia (Ref. 34). Their results showedthat the gain of chromosome 3q defines thetransition from severe dysplasia to invasivecarcinoma.

Spectral karyotyping (SKY)SKY is a novel molecular cytogenetic FISH-basedtechnique that allows colour karyotyping ofhuman and mouse chromosomes (Refs 14,35). Whereas FISH limits analysis to specificchromosomes or regions of chromosomes, andCGH visualises only those changes that result invariations in copy number, SKY permits thevisualisation of all chromosomes at one time,‘painting’ each pair of chromosomes a differentfluorescent colour. Before the development of thistechnique, molecular cytogeneticists analysedchromosomes with staining techniques thatproduced a black-and-white banding pattern.However, the identification of all chromosomalaberrations in a complex karyotype was often notpossible from such patterns. SKY has been appliedto a variety of human malignancies and mousemodel systems (Refs 36, 37), and it has been highlyeffective in deciphering many complex karyotypicrearrangements.



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Figure 3. Comparative genomic hybridisation (CGH) using whole-genomic DNA probes. CGH beginswith the isolation of both genomic DNA from a tumour sample and genomic DNA from an individual who has anormal karyotype (the control DNA). Next, the two genomes are differentially labelled; for example, the controlDNA with the red fluorochrome rhodamine, and the tumour DNA with the green fluorochrome fluoresceinisothiocyanate (FITC). Excess unlabelled human Cot-1 DNA is added to the probe mixtures to suppress therepetitive DNA sequences that are present in both genomes. The differentially labelled genomes are thencombined and hybridised to normal metaphase chromosomes. The relative intensities of the green and redfluorochromes reflect the actual copy-number changes that have occurred in the tumour genome. DNAlosses and gains are indicated by a shift to red (dark grey) and green fluorescence (light grey), respectively(fig003trn).

Addition of Cot-1 DNA

Hybridisation of differentially labelled whole-genomic probesto normal metaphase chromosomes

Isolation and labellingof whole-genomic control

DNA with rhodamine

Isolation and labellingof whole-genomic tumour

DNA with FITC

Expert Reviews in Molecular Medicine 2000 Cambridge University PressComparative genomic hybridisation (CGH) using whole-genomic DNA probes

Loss of DNA shifts colour of region to red

Gain of DNA shifts colour of region to green

(which fluoresces red) (which fluoresces green)

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Figure 4. Comparative genomic hybridisation (CGH) analysis of a lymph node metastasis from a renalcell carcinoma. The metaphase spreads shown in this figure were derived from a patient suffering fromvon Hippel-Lindau syndrome, a hereditary form of renal cell carcinoma. (a) This metaphase spread has beenvisualised using a green fluorochrome that, in this particular sample, is specific for tumour DNA. (b) The samemetaphase spread has been visualised using a red fluorochrome that, in this particular sample, is specific forthe control DNA. (c) This image shows an electronically blended composite of the metaphases shown in parts‘(a)’ and ‘(b)’, which are labelled with green and red fluorochromes, respectively. A chromosomal gain in thetumour is reflected by a stronger intensity of the green fluorescence, whereas a loss is reflected by a strongerintensity of the red fluorescence. (d) This profile shows the ratio of the metaphases that are shown in parts ‘(a)’and ‘(b)’. The most prominent loss can be seen at chromosome 4q, whereas the most prominent gain is atchromosome 10q. The grey boxes in the profile represent chromosomal regions that are rich in heterochromatin,which cannot be interpreted owing to the abundance of highly repetitive DNA (fig004trn).

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Figure 5. Schematic of cytogenetic analysis using spectral karyotyping (SKY) (see next page for legend )(fig005trn).

24 flow-sorted human chromosomes

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1B 1C 2E 3A 3C 3D

2 3 22 X Y

+ Cot-1 DNA

Labelling of the individual chromosome-painting probes using the various combinations of fluorochromes

SKY probe mixture, which contains all of the

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Hybridisation at 37 Cfor 24—72 hours

Detection steps to visualise probes and to remove unbound

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Schematic of cytogenetic analysis using spectral karyotyping (SKY)

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Methodology of SKYSKY is based on the simultaneous hybridisationof 24 chromosome-specific composite probes.Such chromosome-painting probes are generatedfrom flow-sorted human (or mouse) chromosomes(see Fig. 5). Chromosome-specific spectra areproduced by labelling each chromosome libraryeither with a single fluorochrome or with specificcombinations of multiple fluorochromes. Theuse of different combinations of fluorochromesfor DNA labelling increases the number oftargets that can be discerned. A DOP−PCR(degenerate oligonucleotide primer–polymerasechain reaction) labelling reaction is used toincorporate the fluorochromes into the DNA.Using a combinatorial labelling scheme andfive different fluorochromes, 31 differenttargets can be distinguished. The chromosome-painting probes are pooled with an excess ofCot-1 DNA, to suppress the repetitive sequences,and hybridised onto metaphase chromosomepreparations. After incubation for 24–72 hoursat 37°C, detection steps are used to visualisethe fluorochromes, and residual probe isremoved by post-hybridisation washes. Imageacquisition is based on a spectral imaging systemusing an interferometer and a CCD camera.The hybridised sample is illuminated, using acustom-designed triple band-pass optical filter,and the light that is emitted from each pointof the sample is collected with the microscopeobjective. The collected light is transferred to aSagnac interferometer in which an optical path

difference is produced. This is performed at allpixels using a CCD camera. Using Fouriertransformation, the emission spectrum is retrievedand processed by a computer. This process allowsthe spectral signature to be measured at allimage points (pixels). The measurement forms thebasis for chromosome classification by assigningall pixels with identical spectra-unique colours(Refs 38, 39). The technique results in a specificcolour being assigned to each chromosome inthe image (see Fig. 6). One advantage of thesystem is that the image is acquired with a singlefilter set.

SKY as a diagnostic toolThe applications of SKY for identifyingchromosomal aberrations that are involved inhuman disease are manifold. Karyotype analysisbased on chromosomal banding is routinelyperformed in prenatal and postnatal cytogeneticlaboratories. SKY is suitable for the identificationof subtle chromosomal aberrations, such as thetranslocation of telomeric chromatin, which isdifficult to discern using banding alone, andthe identification of small markers, which remainelusive after conventional banding analysis(Refs 40, 41). In a recent study by Schröck andcolleagues, 16 samples that had constitutionalchromosome abnormalities were analysed byGTG banding (also known as Giemsa–trypsin–Giemsa banding or G banding). GTG banding isa technique that uses an enzyme to degrade someof the proteins that bind to the chromosomes.

Figure 5. Schematic of cytogenetic analysis using spectral karyotyping (SKY). (a) First, 24 flow-sortedhuman chromosomes (chromosomes 1–22, X and Y) are combinatorially labelled with at least one, and asmany as five, fluorochrome combinations to create a unique spectral colour for each chromosome pair. Aliquotesof the ‘painted’ chromosomes are pooled together with an excess of Cot-1 DNA. Cot-1 DNA is used to suppressrepetitive sequences in the genome. (b) The SKY probe mixture is then hybridised to a metaphase chromosomepreparation at 37°C for 24–72 hours. (c) Next, detection of the hybridised sample takes place by washing theslide in solutions containing formamide and SSC (sodium chloride, sodium citrate) to remove unboundnucleotides. Incubation with fluorochrome-conjugated antibodies is used to visualise the probes. (d) Visualisationof the SKY hybridisation requires the use of a Spectracube connected to an epifluorescence microscope. All ofthe fluorochrome dyes within the sample are simultaneously excited within a single exposure and are imagedwith a custom-designed triple band-pass optical filter. The light is sent through a Sagnac interferometer thatgenerates an optical path difference for each pixel that is specific for a certain fluorochrome or fluorochromecombination in the image. (e) Fourier transformation is used to recover the spectrum from this interferogram. Aspectral classification is then used to assign a discrete colour to all pixels with identical spectra. The visualisationsoftware applies an RGB (red, green, blue) look-up table to the raw spectral image, resulting in the infra-redemission being displayed in red, the red emission in green and the green emission in blue. The spectralmeasurement can then be converted to a display image that is based on the fluorescence intensities of all ofthe chromosome-painting probes (1); classification colours are also assigned to the chromosomes, based onthe spectra from every pixel (2). This measurement forms the basis for automated chromosome identification.(See also Fig. 6) (fig005trn).



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Figure 6. Spectral image of the chromosomesof the primary bladder tumour cell line RT4. Thisimage shows balanced translocations betweenchromosome 8 and chromosome 17. Spectralkaryotyping (SKY) also reveals that chromosome6 is involved in an unbalanced translocation withchromosome 15. A derivative chromosome 12 thatis involved in an unbalanced translocation withchromosome 1 is also observed. A derivativechromosome refers to a structurally rearrangedchromosome generated from the fusion of two ormore chromosomes or by multiple aberrationswithin a single chromosome. It always refers to thechromosome with the intact centromere. WithoutSKY, these complex rearrangements would bedifficult to detect because the chromosomes thatare involved have similar banding patterns.Abbreviations used: ‘der ’ = derivative; ‘t’ =translocation (fig006trn).

After enzyme treatment with the proteasetrypsin, Giemsa dye selectively binds to the A–Tnucleotides, creating a black-and-white bandingpattern. Using SKY, G-banding diagnosis wasredefined in nine of these cases, revealingtranslocations that were previously unobserved.In five cases, SKY supported the G-bandingresults. As for the remaining two cases, G-bandingresults suggested subtle rearrangements thatwent undetected by SKY. FISH experimentswere conducted with locus-specific probes that

were optimal for detecting submicroscopicintrachromosomal translocations and deletions(Ref. 42). In another study involving SKY,Padilla-Nash and colleagues (Ref. 43) studiedthe bladder cancer cell line BK-10 to identifymultiple structural aberrations: SKY redefined11 aberrations that had previously been detectedby G banding, detected four hidden chromosomalrearrangements and confirmed the presence of12 identified markers.

SKY has also been developed for mousemodels of human cancers (Ref. 35). Weaverand colleagues used SKY to analyse chromosomalaberrations in mammary gland tumours in micethat were carrying the mammary tumour virus(MMTV)–cmyc transgene (Ref. 44). A recurringpattern of chromosome alterations was observed.The loss of chromosome 4 was detected in fiveof the eight tumours. Mouse chromosome 4has regions that are orthologous with region1p31–p36 in humans, which is deleted in somehuman breast carcinomas. Also detected in fourof the tumours were translocations involvingthe distal region of chromosome 11, which ishomologous to human chromosome 17q, whichis where the BRCA1 gene is located. Thesefindings were significant because they showedthat similar chromosomal loci are involved inmouse and human breast tumours, thereforevalidating this mouse model for human carcinomas.

ConclusionsCytogenetic research and diagnostics havegreatly benefited from employing moleculartechniques. In particular, the development ofFISH techniques has widened the plethora ofapplications considerably. The considerable gapin resolution between traditional cytogenetictechniques (megabase pairs) and molecularbiology techniques (base pairs) has been closedto a large extent by molecular cytogenetics, whichallows the assessment of genetic changes onchromosome preparations. The issue of sensitivityhas been successfully tackled by devising newprotocols for the detection of fluorescently taggedprobes as small as 200 base pairs. The thirddefining technical parameter, multiplicity, hasbeen solved by recent developments of colourkaryotyping that allow the visualisation of allhuman or mouse chromosomes simultaneouslyin different colours. One very importantdevelopment in molecular cytogenetic techniqueswas the introduction of CGH, which has proved


Spectral image of the chromosomes ofthe primary bladder tumour cell line RT4

t(8;17)

t(8;17)

der(6)t(6;15)

der(12)t(1;12)

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to be an exceedingly valuable tool for thecharacterisation of chromosomal imbalances insolid tumours. Together, these developmentshave contributed to the refinement of thefield of molecular cytogenetics and have, in anunpredictable way, increased the flexibility withwhich experiments can be designed. Thus, theapplications of these techniques have beenextended beyond the mere diagnosis ofchromosomal abberrations to functional andcomparative basic research.

AcknowledgementsNicole McNeil would like to thank: the entireRied laboratory, past and present for theircontinued support; Dr Hesed Padilla-Nash forher contribution of the SKY image; Dr JohnPhillips for his contribution of the CGH image;and Dr Padilla-Nash, Dr Eva Hilgenfeld andMrs Turid Knutsen for their helpful critique ofthis article.

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Citation details for this article

Nicole McNeil and Thomas Ried (2000) Novel molecular cytogenetic techniques for identifying complexchromosomal rearrangements: technology and applications in molecular medicine. Exp. Rev. Mol. Med. 14September, http://www-ermm.cbcu.cam.ac.uk/00001940h.htm



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Features associated with this article

FiguresFigure 1. Examples of different types of fluorescence in situ hybridisation (FISH) probes (fig001trn).Figure 2. Fluorescence in situ hybridisation (FISH) using whole-chromosome-painting probes (fig002trn).Figure 3. Comparative genomic hybridisation (CGH) using whole-genomic DNA probes (fig003trn).Figure 4. Comparative genomic hybridisation (CGH) analysis of a lymph node metastasis from a renal cell

carcinoma (fig004trn).Figure 5. Schematic of cytogenetic analysis using spectral karyotyping (SKY) (fig005trn).Figure 6. Spectral image of the chromosomes of the primary bladder tumour cell line RT4 (fig006trn).

Further reading, resources and contacts

The following publications provide a useful source of background information on cytogenetic methodologyand molecular cytogenetics and their applications.

Heim, S. and Mitelman, F. (1995) Cancer Cytogenetics, Wiley Liss

Knutsen, T. and Ried, T. (2000) SKY: A Comprehensive Diagnostic and Research Tool: A Review of the First300 Published Cases. J Assoc Genet Technol 26 (1), 3-15

Mitelman, F. (1994) Catalog of Chromosomal Aberrations in Cancer (5th edn), Wiley-Liss

Mitelman, F., ed. (1995) An International System for Human Cytogenetic Nomenclature (ISCN), Karger, NewYork, USA

Rooney, D.E. and Czepulkowski, B.H., eds (1992) Human Cytogenetics: A Practical Approach, Vol. I and II,Oxford University Press

Sandberg, A. (1990) The Chromosomes in Human Cancer and Leukemia, Elsevier Science

Veldman, T. et al. (1997) Spectral Karyotyping. In The AGT Cytogenetics Laboratory Manual (3rd edn)(Barch, M.J. et al., eds), pp. 591-595, Lippencott-Raven Publishers

Verma, R.S. and Babu, A. (1995) Human Chromosomes: Principles and Techniques, McGraw-Hill

The following companies may be contacted for hardware and software as well as for information oncytogenetics applications.

Applied Spectral Imaging Inc., USA (2120 Las Palmas Drive, Suite D, Carlsbad, CA 92009, USA; Tel: +1760 929 2840; Fax: +1 760 929 2842; E-mail: [email protected])

Applied Spectral Imaging, Europe (Dr Michael Koehler, Neurottstr. 12D-68535, Edingen-Neckarhausen,Germany; Tel: +49 6203 923800; Fax: +49 6203 923826; E-mail: michael.koehler@spectral–imaging.com)

Chroma Technology Corp. (72 Cotton Mill Hill, Unit A9, Brattleboro, VT 05301, USA; Tel: +1 800 824 7662or +1 802 257 1800; Fax: +1 802 257 9400; E-mail: [email protected])

http://www.chroma.com

Leica Microsystems (Imaging Solutions Ltd, Clifton Road, Cambridge, CB1 3QH, UK; Tel: +44 1223 411411;Fax: +44 1223 412526; E-mail: [email protected])

http://www.leica.co.uk/ia/homepage.htm

Vysis, Inc. (3100 Woodcreek Drive, Downers Grove, IL 60515-5400, USA; Tel: +1 800 553 7042 or+1 630 271 7000; Fax: +1 630 271 7138; E-mail: vysis_help@vysis .com)

http://www.vysis.com

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