j reverse method aberrant cytogenetics

J Med Genet 1992; 29: 299-307

Reverse chromosome painting: a method for therapid analysis of aberrant chromosomes inclinical cytogenetics

N P Carter, M A Ferguson-Smith, M T Perryman, H Telenius, A H Pelmear,M A Leversha, M T Glancy, S L Wood, K Cook, H M Dyson,M E Ferguson-Smith, L R Willatt

Department ofPathology, Universityof Cambridge, TennisCourt Road,Cambridge CB2 lQP.N P CarterM A Ferguson-SmithM T PerrymanA H PelmearM A Leversha

East Anglian RegionalGenetics Service,Addenbrooke'sHospital, Hills Road,Cambridge.M A Ferguson-SmithM T GlancyS L WoodK CookH M DysonM E Ferguson-SmithL R Willatt

CRC Human CancerGenetics ResearchGroup, Department ofPathology, Universityof Cambridge, andDepartment ofClinical Genetics,Karolinska Hospital,Stockholm, Sweden.H Telenius

Correspondence toDr Carter.

Received 9 December 1991.Accepted 6 January 1992.

AbstractWe describe a method, termed reverse

chromosome painting, which allows therapid analysis of the content and break-points of aberrant chromosomes. Themethod involves the sorting of smallnumbers of the aberrant chromosomefrom short term blood culture prepara-

tions or cell lines by using bivariate flowkaryotype analysis. The sorted chromo-somes are amplified and biotin labelledenzymatically using a degenerate oligo-nucleotide-primed polymerase chainreaction (DOP-PCR), the product annealedto metaphase spreads from normal sub-jects, and hybridisation detected usingfluorescence in situ hybridisation (FISH).We show the usefulness of this methodfor routine clinical cytogenetics by theanalysis of cases involving an insertion, a

deletion, a translocation, and two cases ofa chromosome with additional materialof unknown origin. The method has par-

ticular application for the rapid resolu-tion of the origin of de novo unbalancedchromosome duplications.

The development of chromosomal in situ sup-

pression (CISS) hybridisation with flow sortedchromosome libraries and the detection of sig-nals by non-isotopic meansl4 has provided a

powerful tool for the rapid analysis of humanchromosome aberrations. The use of thesetechniques, termed chromosome painting, isbecoming increasingly widespread in the rou-

tine clinical cytogenetics laboratory.56In conventional chromosome painting, a

typical investigation would involve initialbanding studies for the identification of abnor-mal chromosomes followed by painting of thepatient's metaphase spreads with the appropri-ate chromosome library or libraries. Thisstrategy works efficiently for confirmation andrefinement of the diagnosis of abnormal karyo-types in cases where banding analysis can

provide an indication of which chromosomelibraries to use. However, for chromosomeswith small rearrangements or chromosomescontaining additional chromosomal material ofunknown origin, the only approach is to tryeach library in turn until hybridisation of theabnormal chromosome is observed.6 An addi-tional limitation of the traditional chromosomepainting approach in such cases is that sub-

chromosomal region information about thesource of the additional material is notobtained. Furthermore, deletions cannot bevisualised by hybridisation of chromosomelibraries onto the abnormal chromosome.

In this paper we describe a reverse chromo-some painting technique in which the probe isgenerated from the aberrant chromosome itselfand applied to metaphase spreads of normalsubjects using fluorescence in situ hybridisa-tion (FISH), so highlighting the constituentsof the aberrant chromosome directly on nor-mal chromosomes. Briefly, small numbers ofthe aberrant chromosome are flow sorted froma routine peripheral blood culture and ampli-fied in a general way using a degenerate oligo-nucleotide primed polymerase chain reaction(DOP-PCR) protocol.7 This complex probe isbiotinylated during secondary DOP-PCRcycles and then hybridised onto normalmetaphase spreads using CISS. Detection byavidin-fluorescein isothiocyanate conjugatesenables chromosome regions present in theaberrant chromosome to be visualisd on thenormal metaphase chromosomes by usingfluorescence microscopy. Recently we haveshown that this approach can be used for theanalysis of the breakpoints of translocatedchromosomes sorted from cell lines.7 In thispaper, we show that the reverse chromosomepainting approach is readily applicable to clin-ical cytogenetic practice, without the need forestablishment of cell lines, by reporting theanalysis of five diagnostic cases involving aninsertion, a deletion, a translocation, and twocases with additional chromosomal material ofunknown origin.

MethodsCELL PREPARATIONSPhytohaemagglutinin stimulated peripheralblood cultures (05 ml of blood in 8 mlmedium) were established and metaphasespreads were prepared using standard tech-niques with overnight synchronisation usingmethotrexate and release with bromodeoxyur-idine.8 Routine cytogenetic analysis was per-formed on GTL banded preparations. Slidesprepared for in situ hybridisation were allowedto air dry and then fixed further in methanol:acetic acid (3:1) for one hour. These slideswere then dehydrated through a 70%, 90%,and 100% ethanol series, fixed in acetone for10 minutes, and allowed to air dry. Slides were

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Carter, Ferguson-Smith, Perryman, Telenius, Pelmear, Leversha, Glancy, Wood, Cook, Dyson, Ferguson-Smith, Willatt

stored desiccated at room temperature forbetween seven and 14 days before use.For flow analysis, one or two blood cultures

as above were blocked after 72 or 96 hours with0-1 gtg/ml colcemid (BRL) overnight. Leuco-cytes were separated by centrifugation (800 gfor 20 minutes) over a density step gradient ofLymphopaque 1119 (Sigma). Cells at thedensity interface were removed, pooled ifnecessary, and washed twice in 20 ml ofmedium (RPMI 1640, 16% fetal calf serum,2 mmol/l L-glutamine, penicillin 100 units/ml,streptomycin 100 mg/ml, colcemid 01 Ipg/ml)by centrifugation (200 g for eight minutes),resuspended in 2 ml of hypotonic buffer(75 mmol/l KC1, 0 2 mmol/l spermine,0 5 mmol/l spermidine), and allowed to swellfor 15 minutes at room temperature. Aftercentrifugation for five minutes at 400 g, the cellpellet was resuspended in 380 gl of polyaminebuffer (15 mmol/I tris(hydroxymethyl)methy-lamine, 0 2 mmol/l spermine, 0 5 mmol/l sper-midine, 2 mmol/l EDTA, 0-5 mmol/l EGTA,80 mmol/I KC1, 20 mmol/I NaCl, 14 mmol/IP-mercaptoethanol, 0-25% Triton-X 100, pH7-2), and incubated on ice for 10 minutes. Thesuspension was then vortexed for 10 to 15seconds to release the chromosomes into sus-pension. The chromosomes were stained bythe addition of 40 gg/ml of Chromomycin A3,2 mmol/l magnesium sulphate, and 2 gtg/ml ofHoechst 33258 and incubated for at least twohours. Ten minutes before flow analysis,sodium citrate and sodium sulphite wereadded to a final concentration of 10 mmol/l and25 mmol/l respectively to give a final suspen-sion volume of 500 gl.

FLOW CYTOMETRYChromosome preparations were analysed on aFACStar Plus flow sorter (Becton Dickinson)equipped with two 5W argon ion lasers asdescribed previously.9 Between 300 and 400specific chromosomes were sorted directly into500 p1 PCR tubes containing 33 pl of purewater and then stored at 4°C.

DOP-PCR AMPLIFICATIONDOP-PCR amplification from the sorted chro-mosomes was performed as described pre-viously.7 Briefly, reaction buffer (25 mmol/lN-tris (hydroxymethyl) methyl-3-amino-pro-anesulfonic acid, 50 mmol/l KCI, 2 mmol/lMgCl2, 1 mmol/l dithiothreitol, pH 90), de-tergent (005% polyoxyethylene ether W-1),dinucleotide triphosphates (200 jimol/l ofeach triphosphate), primer 6-MW (5' CCGACT CGA GNN NNN NAT GTG G 3'where N = any base, 2 jmol/l) and 2-5 units ofTaq polymerase (NBS Biologicals) were addedfrom concentrated stocks to give a final reac-tion volume of 50,l, which was overlayeredwith 30 p1 of mineral oil. After an initial de-naturation for nine minutes at 94°C, five cyclesof 94°C for one minute, 30°C for 1.5 minutes,transition at 0 23°C/sec to 72°C held for threeminutes were followed immediately by 35cycles of 94°C for one minute, 62°C for oneminute, and 72°C for three minutes. The finalextension at 72°C was increased to 10 minutes.Biotinylation was achieved by subjecting300ng of primary PCR product in reactionbuffer as above but supplemented with dUTP-1 1-biotin (0-28 mmol/l final concentration,

Figure 1 Localisation of hybridisation signal on fluorescent reverse banded chromosomes (normal chromosomes,paint from case 3). (A) Image from detector 1 (propidium iodide fluorescence) of the confocal microscope showingreverse bands. (B) Image from detector 2 (fluorescein isothiocyanate fluorescence) merged with image from detector1 showing localisation of hybridisation from q31.2-.qter on chromosome 7.

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Reverse chromosome painting: a method for the rapid analysis of aberrant chromosomes in clinical cytogenetics

Sigma) to further amplification (initial de-naturation at 94°C for three minutes followedby 25 cycles of 94°C for one minute, 62°C for 2minutes, and 72°C for three minutes, finalextension at 72°C increased to 10 minutes). AllPCR reactions were performed on a Trio ther-mal cycler (Biometra) and product concentra-tion was determined using a TKO 100 DNAfluorometer (Hoefer).

CISS HYBRIDISATION AND PROBE DETECTIONHybridisations and detection were carried outusing a modification of the procedure de-scribed by Pinkel et al.4 Briefly, for each slide,50 ng of probe and 500 ng Cot 1 DNA (BRL)were made up to 20 g±l with hybridisationbuffer'0 (50% v/v deionised formamide, 10%w/v dextran sulphate, 2 x SSC, 40 mmol/l

Case 1

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sodium phosphate, 01% SDS, 1 x Denhardt'ssolution, pH 7-0. 1 x Denhardt's solution=0-02% polyvinylpyrrolidone, 0-02% Ficoll,0-02% bovine serum albumin. 1 x SSC=0 15 mol/l NaCl, 15 mmol/l sodium citrate, pH7 5), mixed well, denatured for 10 minutes at65°C, and preannealed for one hour at 37°C.Slides were denatured in 70% formamide,2 x SSC for exactly two minutes at 65°C,quenched in ice cold 70% ethanol, dehydratedthrough a 70%, 90%, and 100% ethanol series,and air dried. The preannealed probe wasapplied to slides and allowed to hybridiseovernight at 42°C. After stringency washes in50% formamide, 1 x SSC, hybridisation wasdetected by incubation with avidin-FITCDCS (Vector Labs). The signal was usuallyamplified by further incubation with bio-tinylated anti-avidin (Vector Labs) followed

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Figure 2 Partial karyotypes by GTL banding. Bars indicate approximate breakpoints. The abnormal chromosomeis on the right of each pair. For detailed explanation of each case refer to text.

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by avidin-FITC. Chromosomes were counter-stained and reverse banded by mounting theslides in Citifluor antifade AFl (Citifluor Ltd)containing 2 5 .ig/ml of DAPI and 0-51g/mlof propidium iodide. Hybridised slides wereassessed using a Nikon Optiphot fluorescencemicroscope with a 100 x, 1-4 na objective andan I2 filter block. Images were recorded asgrey levels using both detectors (detector 1,500 to 560 nm; detector 2 <600 nm) of anMRC 600 confocal scanning head (Biorad) anddisplayed merged in pseudocolour (FITCfluorescence in green, PI fluorescence in red).The band location of FITC signal was deter-mined by toggling the FITC signal on and offto allow the banding pattern beneath the signalto be displayed (fig 1).

ResultsCASE 1The patient was a 33 year old female, para 2 + 2(the two fetal losses were terminations of preg-nancies with neural tube defects). The first liveborn was a normal male; however, the secondwas a male with unilateral coloboma of the irisand choroid, a possible midline brain defectidentified on scan, and hypotonia.Chromosome analysis of cultured lympho-

cytes from the second male child showed anabnormal karyotype, 46,XY, 13q-. Maternalchromosomes showed an interstitial deletion/insertion involving chromosomes 1 and 13,with a segment around band q33 on the longarm of chromosome 13 inserted into p36 onthe short arm of chromosome 1 (fig 2). Thekaryotype was interpreted as 46,XX,ins(1;13)(p36;q32q34). It was noted that band

13q33 appeared to resolve into two bandswhen inserted into chromosome 1 and variousinterpretations were considered.From the maternal flow karyotype (fig 3), it

can be seen that the chromosome 13 with thedeletion is readily resolved but that the chro-mosome 1 with the insertion could not beseparated from the normal chromosome 1.Therefore, it was necessary to sort both ofthese chromosomes 1 to produce the paintinvolving the inserted chromosome. A typicalnormal male metaphase hybridised with thispaint is shown in fig 4. In addition to the twonormal chromosomes 1, signal was detectedon the graphics display from distal 13q31,through 13q32, and involving a large part of13q33. These breakpoints were confirmed byanalysis of the paint derived from the chromo-some with the deletion (data not shown). Theorigin and position of the insertion was con-firmed when a normal chromosome 13 paintwas hybridised to the patient's chromosomes(data not shown).

CASE 2The proband, a 1 year old girl, was referred forroutine cytogenetic investigation from thelocal Child Development Centre. She hadsmall hands, unusual facies, and general de-velopmental delay. Chromosome analysis ofcultured lymphocytes showed a female karyo-type with an interstitial deletion in thelong arm of one chromosome 16 involvingq13 to q21 (karyotype 46,XX,del(16)(pter-+ql3::q21-+qter)). The small G dark bandpresent between q12 and q21 on the derivativechromosomes (fig 2) can only be explained bybreakpoints distal in 16q13 and distal in16q21. Parental karyotypes were apparentlynormal.

Despite a poor yield of cells and a lowmitotic index resulting in a flow karyotype ofreduced resolution (fig 5), the deleted chromo-some 16 could be distinguished easily and wassorted for PCR amplification. A typical normalmetaphase hybridised with the derivative paintis shown in fig 4B. While most of the length ofthe chromosomes 16 is painted, signal is miss-ing from the centromere and the region involv-ing distal 16q13 and all of 16q21.

CASE 3A 33 year old expectant mother (para 0+ 1)was referred for prenatal diagnosis by chorio-nic villus sampling (CVS) at 11 weeks' gesta-tion. Her partner's karyotype showed anapparently balanced reciprocal translocation,46,XY,t(7;21)(q31 ;q22), identified through anextensive family study of recurrent miscar-riages. The CVS analysis showed the fetus tobe female with the apparently balanced recip-rocal translocation, 46,XX,t(7;21)(q31 ;q22).At term a phenotypically normal female babywas born and the karyotype was confirmed bychromosome analysis from a cord bloodsample taken at delivery (fig 2).The flow karyotype of the child is shown in

fig 6. The t(7;21) is confirmed by half the

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Figure 3 Maternal flow karyotype from case 1 (see text for details). The peakcontaining the normal and derivative chromosomes 1 is indicated.

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303Reverse chromosome painting: a method for the rapid analysis of aberrant chromosomes in clinical cytogenetics

Figure 4 Reverse and conventional chromosome painting analyses of cases 1 to 4 (see text for details). (A)Chromosome paint derivedfrom the normal and derivative chromosomes I in case I hybridised onto a normalmetaphase spread. The chromosome 13 material present in the derivative chromosome 1 is visualised on the twonormal chromosomes 13 (arrowed). (B) Chromosome paint derived from the chromosome 16 with the deletion in case2 hybridised onto a normal metaphase spread. The region of chromosome 16 deleted in the patient is represented bythe region on the long arms of the normal chromosomes 16 which are not painted (arrowed). (C) Chromosome paintderivedfrom the derivative chromosome 21 in case 3 hybridised onto a normal metaphase spread. The regions ofchromosomes 7 and 21 involved in the translocation are visualised directly. (D) Chromosome paint from the normalchromosome 21 in case 4 hybridised onto a normal metaphase spread. Only the two normal chromosomes 21 arepainted. (E) Chromosome paint from the derivative chromosome 21 in case 4 hybridised onto a normal metaphasespread. As well as signals on the two normal chromosomes 21 (arrowed) and acrocentric short arms, regionq32.1 -+qter of the normal chromosomes 14 are painted. (F) Chromosome paint from sorted normal chromosome 14hybridised onto a metaphase spread of the patient in case 4. As well as the two chromosomes 14, the distal part of theshort arm of the derivative chromosome 21 is painted.

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Figure 5 Flow karyotype from case 2. The peaks representing the normal andderivative chromosomes 16 are indicated.

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Chromomycin A3 fluorescenceFigure 6 Flow karyotype from case 3. The peaks representing the normal andderivative chromosomes 7 and 21 are indicated.

normal number of events in the chromosome 7and chromosome 21 peaks. The derivative 21is resolved clearly just below chromosome 18while the derivative 7 is contained within thechromosome 9 to 12 cluster. Therefore onlythe derivative 21 could be sorted for PCRamplification. A typical normal male meta-

phase hybridised with the derivative 21 paintis shown in fig 4C. The two chromosomes7 show signal from q31.2--qter, while thetwo chromosomes 21 show signal fromqi 1 .2-*q22.2.

CASE 4The proband, a 1 year old boy, was referred forroutine cytogenetic investigation from thelocal outpatient department. He had hypotoniaand delayed motor development. Chromosomeanalysis of cultured lymphoctyes showed amale karyotype, with additional material onthe short arm of one chromosome 21 (karyo-type 46,XY,21p +, fig 2). As the short arm ofthe aberrant chromosome 21 was AgNOR andC band negative, we were unable to determinethe origin of the additional euchromatic mater-ial using the conventional cytogenetic ap-proach. Parental karyotypes were normal, con-firming that this unbalanced chromosomeduplication had occurred de novo.The flow karyotype of this boy is shown in

fig 7. As well as heteromorphisms of chromo-somes 13, 20, and 22, the normal chromosome21 and the derivative chromosome 21 areresolved clearly. Both the normal chromosome21 and derivative chromosome 21 were sortedseparately for amplification by PCR. The paintgenerated from the normal chromosome 21showed hybridisation only to chromosomes 21on a normal male metaphase spread (fig 4D).However, the derivative chromosome 21 paintshowed hybridisation on normal metaphasespreads to chromosomes 21, acrocentric shortarms, and to the region q32. 1--qter of chro-mosomes 14 (fig 4E). From this analysis thechild has a duplication of chromosome 14involving region q32.1 -+qter. In this case thediagnosis of chromosome 14 as the source ofthe additional material in the derivative chro-mosome 21 was confirmed by hybridisingmetaphases from the child with a DOP-PCRpaint generated from normal chromosomes 14sorted from a lymphoblastoid cell line. Fig 4Fshows an example of this conventional paint-ing analysis where both chromosomes 14 andthe distal part of the short arm of one chromo-some 21 display fluorescent signal.

CASE 5The patient, a 6 year old girl tall for her age,was referred for investigation because ofdevelopmental delay and mild dysmorphicfeatures. Chromosome analysis of culturedlymphocytes (fig 2) showed a female karyotypewith additional material on the short arm ofchromosome 8 distal to 8p22 (karyotype46,XX,8p+). As a result of the addition, theregion 8p22-pter was estimated to be approx-imately twice the normal size. However, thechromosomal origin of the extra material couldnot be determined by GTL banding. Bothparents have apparently normal karyotypes.The flow karyotype of the patient is shown

in fig 8. The derivative chromosome 8 wasfound to lie between chromosomes X and 7 onthe flow karyotype. A small sorting gate was

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Figure 7 Flow karyotype from case 4. The peaks representing the normal andderivative chromosomes 21 are indicated.

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Chromomycin A3 fluorescenceFigure 8 Flow karyotype from case S. The peaks representing the normalchromosomes X, 8, and 7, and the derivative chromosome 8 are indicated.

set around this region in an attempt to includeas little of chromosomes X and 7 as possiblebut contamination with these chromosomeswas inevitable. The paint generated from thissort shows strong hybridisation to the chromo-somes 8 on metaphase spreads of a normal

male (fig 9A) with weaker hybridisation tochromosomes X and 7. In particular, there wasno evidence of incomplete painting of thenormal chromosomes 8 as might be expected ifthe abnormal chromosome 8 from the patientwas the result of a reciprocal translocation. Ashybridisation to chromosomes X and 7 wasexpected from the impure nature of the flowsorting and as no signals other than those onchromosomes 8 were detected, we concludedthat the derivative chromosome 8 was theresult of a duplication of chromosome 8. Thisconclusion was tested by hybridising thepatient's metaphases with a normal chromo-some 8 paint. It can be seen from fig 9B thatthe entire length of both the normal and theaberrant chromosomes 8 are painted, so con-firming the intrachromosomal origin of theaditional chromosomal material.

DiscussionIn this study we have shown that reversechromosome painting, where the paint isgenerated rapidly from small numbers of flowsorted aberrant chromosomes and applied tonormal metaphase spreads, is a useful tech-nique for the analysis of abnormal karyotypes.Reverse chromosome painting is clearly themethod of choice for determining the origin ofde novo unbalanced chromosome duplications(for example cases 4 and 5) which cannot beresolved by conventional banding techniques.It is encouraging to note that in all but onecase, the specific band assignments found bycytogenetic analysis and the reverse paintingtechnique were consistent.

In case 1, cytogenetic analysis indicated aninsertion of 13q33 into lp36 although theprecise location of the insertion into 1p36could not be determined by conventionalbanding. The reverse painting showed theinvolvement of a region including part of distal13q31, all of 13q32, and most of 13q33. Allow-ing for the modification of G band staining atthe site of translocation breakpoints, the inser-tion of this region above lp35 is consistentwith the banding pattern of the derivativechromosomes in that an enlarged G dark bandat 1p35 (lp35 plus 13q31) would be producedwith an additional G dark band derived fromthe distal part of 13q33 present below lp36.1(fig 2). Thus, the consensus karyotype for thissubject becomes 46,XX,dir ins(1;13)(1pter-+Ip36.1::13q33-+ 13q31::lp36.1 - qter;13pter13q31 ::13q33- 13qter).In case 3, cytogenetic analysis of the trans-

location showed breakpoints at bands 7q31and 21q22, but more detailed analysis was notpossible as we were unable to determine, evenat high banding resolution, whether 21q22.2was translocated. However, it was clear fromthe reverse painting that the breakpoints were7q31.2 and distal 21q22.2.

Analysis of case 4 showed immediately thatthe additional chromosomal material on 21pwas derived from the region q32. 1-- qter ofchromosome 14. This result shows the powerof the reverse chromosome painting technique

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Figure 9 Chromosome painting analysis of case 5. (A) Chromosome paint derivedfrom the flow sorting regionbetween chromosomes X and 7 (to include the derivative chromosome 8) in case 5 hybridised onto a normalmetaphase spread. Strong signals can be seen on the normal chromosomes 8 with weaker signals on chromosomes Xand 7. (B) Chromosome paint from sorted normal chromosome 8 hybridised onto a metaphase spread of the patientin case 5. The entire length of the derivative chromosome 8 is painted confirming the chromosome 8 origin of theduplication.

for cases of this type as both the chromosomalorigin and subchromosomal localisation of thede novo duplication is visualised directly.Conventional hybridisation onto the patient'schromosomes with a normal chromosome 14paint (as in fig 4F) indicated that this addi-tional material was indeed derived from chro-mosome 14, but cannot provide information asto which region of chromosome 14 wasinvolved. In hindsight, it can be seen in fig 2that the small G dark band in the short arm ofthe derivative chromosome 21 (which did notsilver stain) is consistent with the presence ofband 14q32.2.The diagnosis of an intrachromosomal

duplication was made by reverse painting incase 5 where, once again, the source of theadditional material arising de novo on 8p wasnot evident on conventional banding. In thiscase, while the chromosome 8 origin of theduplication is clear, it is not possible to deter-mine the regional localisation of the duplica-tion by reverse painting. Regional chromo-some paints made from sorting appropriatetranslocation derivatives should help to resolvethis difficulty. Once the duplication is local-ised, chromosome 8 specific probes derivedfrom the region could be used to define itsextent more accurately. Nevertheless, this caseshows the value of the method for the initialdetection of intrachromosomal aberrations.Only in case 2 was there slight disagreement

in breakpoint assignment between the twoanalysis techniques. The GTL banding pat-tern on the long arm of the derivative chromo-some shown in fig 2 can only be explained bythe loss of distal 16q13 and most but not all of16q21. While reverse painting showed that theproximal end of the deletion occurred distal in

16q13 in agreement with banding analysis,all of band 16q21 appeared to be lost. Oneexplanation for this discrepancy could bethe difference between band positions ofGTL banded and RBF banded preparations.Alternatively, although the visualisation ofbreakpoints using the reverse banding tech-nique appears to be remarkably specific evenon relatively short chromosomes, the fluores-cence complex itself lies above the chromo-some and some positional error is likely tooccur. The amount of complex formed, whichis dependent on probe and detection reagentconcentrations, will determine the degree ofspreading of the signal around the hybridisa-tion site. Another important factor is the set-ting of the gain and threshold of the detectionhardware, as it is possible by using inappro-priate settings for fluorescence signal to spreadout from the specific hybridisation site. Thesefactors, while controlled in this study, can alllead to a slight overestimation of the length of apainted section of a chromosome and thus mayprovide inaccurate breakpoint analysis unlessthis point is appreciated.Some other limitations to the usefulness of

the reverse chromosome painting techniqueshould be mentioned. The DOP-PCR ampli-fication, although producing relatively evensignal on the euchromatin of chromosomes,often fails to paint highly repetitive sequencesin acrocentric short arms, at the centromere,and in some heterochromatic regions. This canbe seen in fig 4 where centromeres and in somecases acrocentric short arms are not painted.This effect is either because of the failure ofthese sequences to amplify (the DOP-PCRamplification is specific for the most 3' six basesequence) (Telenius et al, in preparation) or,

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more likely, because of complete suppressionof the signal by the Cot 1 DNA. However,whether these repetitive regions hybridise ornot is unpredictable and varies from chromo-some amplification to chromosome amplifica-tion. Thus, in case 3, the paint produced fromthe amplified derivative chromosome 21(which contains 21p) did not hybridise to 21pon the normal chromosomes. However, in case4, sequences from 21p were amplified from the21p + chromosome so that the short arms ofthe normal chromosomes 21 were painted. Afurther complication in this latter case is thatacrocentric short arms share the same repetit-ive sequences so that the generated paint willhybridise not only to 21p but also to the otheracrocentric short arms. This is particularlynoticeable in fig 4D. This complication in thevariable amplification of such repetitive chro-mosome regions must be taken into account inthe interpretation of reverse painting analyses.Another consideration is the accuracy and

purity of the initial chromosome sorting fromwhich the paint is generated. When thederivative chromosome is resolved on the flowkaryotype away from the other normal chro-mosomes, a paint specific for the aberrantchromosome can be produced and a straight-forward analysis performed. However, in somecases (for example cases 1 and 5), the deriva-tive chromosomes will overlie- others or be soclose as to preclude pure sorting. However, inthis event, it is still possible to sort the regioncontaining the aberrant chromosome andgenerate a complex paint of both the aberrantand co-sorting normal chromosomes. Mostoften, the co-sorting normal chromosome willnot be involved in the derivation of the aber-rant chromosome and a reverse painting analy-sis will still be possible (for example, cases 1and 5), but care in the interpretation of suchcases must be exercised. Another limitation isfound in the analysis of insertions. Reversechromosome painting can readily identify thechromosome and region of the materialinvolved in the insertion, but cannot identifywhere in the derivative chromosome the inser-tion has taken place. Here conventional paint-ing, using a flow sorted library or DOP-PCRproduct specific for the donor chromosometype, can be used to identify on the insertedchromosome itself the location of the insertion.

It should be mentioned that, while the re-verse painting technique is readily applied tolong term cultures (and we have had particularsuccess with immortalised lymphoblastoid celllines), our aim here is to show the applicationof the method to routine short term culturesfrom peripheral blood samples. Althoughfluorescence activated chromosome sortingmay not be readily available to all chromosomediagnostic laboratories, a cost effective chro-mosome sorting service could be establishedby a small number of supraregional laborator-ies. Only a comparatively small proportion ofabnormal cases encountered by a diagnostic

laboratory would be appropriate for reversepainting.As shown here, the technique is most useful

for determining the nature of de novo unbal-anced chromosome duplications and for deter-mining the extent of deletions. Our experiencewith small marker chromosomes is limited atpresent to two cases with mosaicism for a tinymarker chromosome less than one third thesize ofchromosome 21. In both cases, the extrachromosome did not produce a distinctivepeak on the flow karyotype made from peri-pheral blood cultures and so could not besorted. We do not envisage such problemswith larger marker chromosomes as these havebeen resolved previously using lympho-blastoid cell lines." In fact, reverse paintingmay well be the method of choice in such cases.The reverse painting technique shows pro-

mise in the interpretation of high resolutionbanding analysis where a particular patternmay be explained by several different com-binations of breakpoints'2 (for example, case1). Altogether, the combined techniques ofhigh resolution banding, reverse painting, andconventional painting provide a powerful re-source for diagnostic cytogenetics in the fu-ture.

This work was carried out with supportfrom the Medical Research Council. We aregrateful to G van den Engh for the programANALIST which was used to produce theflow karyotype contour plots.

1 Cremer T, Lichter P, Borden J, et al. Detection of chromo-some aberrations in metaphase and interphase tumourcells by in-situ hybridization using chromosome-specificlibrary probes. Hum Genet 1988;80:235-46.

2 Lichter P, Cremer T, Borden J, et al. Delineation ofindividual human chromosomes in metaphase and inter-phase cells by in-situ suppression hybridization usingrecombinant DNA libraries. Hum Genet 1988;80:224-34.

3 Lichter P, Cremer T, Chang Tang CJ, et al. Rapid detec-tion of chromosome 21 aberrations by in-situ hybridiza-tion. Proc Natl Acad Sci USA 1988;85:9664-8.

4 Pinkel D, Landegent J, Collins C, et al. Fluorescence in situhybridization with human chromosome-specific libraries:detection of trisomy 21 and translocations of chromosome4. Proc Natl Acad Sci USA 1988;85:9138-42.

5 Jauch A, Oaumer C, Lichter P, et al. Chromosomal in-situsuppresson hybridization of human gonosomes and auto-somes and its use in clinical cytogenetics. Hum Genet1990;85:145-50.

6 Hulten MA, Gould CP, Goldman ASH, et al. Chromosomein situ suppression hybridisation in clinical cytogenetics.J Med Genet 1991;28:577-82.

7 Telenius H, Pelmear AH, Tunnacliffe A, et al. Cytogeneticanalysis by chromosome painting using DOP-PCR amp-lified flow-sorted chromosomes. Genes, Chromosomes andCancer 1992;4:257-63.

8 Rooney DE, Czepulkowski BH. Human cytogenetics: a prac-tical approach. Oxford: IRL Press, 1986.

9 Carter NP, Ferguson-Smith ME, Affara NA, et al. Study ofX chromosome abnormality in XX males using bivariateflow karyotype analysis and flow sorted dot blots. Cyto-metry 1990;11:202-7.

10 Viegas-Pequignot E, Dutrillaux B, Magdelenat H, et al.Mapping of single-copy DNA sequences on human chro-mosomes by in situ hybridisation with biotinylatedprobes: enhancement of detection sensitivity by intensi-fied-fluorescence digital-imaging microscopy. Proc NattAcad Sci USA 1989;86:582-6.

11 Waters JJ, Ferguson-Smith ME, Carter NP, et al. Prenataldiagnosis of a double bisatellited marker with an unusualcopy number ratio. Prenat Diagn 1990;10:677-81.

12 Savage JRK. Assignment of aberration breakpoints inbanded chromosomes. Nature 1977;270:513-4.

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