@prefix vivo: . @prefix edm: . @prefix ns0: . @prefix dcterms: . @prefix dc: . @prefix skos: . vivo:departmentOrSchool "Medicine, Faculty of"@en, "Medical Genetics, Department of"@en ; edm:dataProvider "DSpace"@en ; ns0:degreeCampus "UBCV"@en ; dcterms:creator "Henderson, Karen Gwen"@en ; dcterms:issued "2008-12-24T18:53:10Z"@en, "1992"@en ; vivo:relatedDegree "Master of Science - MSc"@en ; ns0:degreeGrantor "University of British Columbia"@en ; dcterms:description """A. de novo chromosomal aberration in a female with severe mental retardation and dysmorphic features has been characterized cytogenetically (Dill et al. 1987). The patient’s karyotype was described as 46,XX,inv dup(8)(p12→23.1). Previous Southern blot dosage analysis of the patient’ s DNA with a probe from the D8S7 locus, which maps to 8p23→8pter (Wood et al. 1986), demonstrated that the patient was monosomic for this locus. This dosage abnormality was interpreted as a consequence of the chromosomal rearrangement, suggesting that the aberrant chromosome was a duplication deficiency chromosome. We have reinvestigated this patient using fluorescent in situ hybridization using cosmids from a flow sorted chromosome 8 library as well as an 8p painting probe mixture generated by Mu element mediated PCR. Both the normal and the inversion duplication chromosome p arms are uniformly labelled by the 8p painting probe mixture. Hybridization of a cosmid from the D8S7 locus results in a hybridization signal on the normal chromosome 8 and a complete lack of signal on the inversion duplication chromosome 8. Hybridization of a cosmid from the D8S133 locus, localized to 8p21→8cen using a hybrid cell panel (Wagner et al. 1991), provides a single hybridization signal on the normal chromosome 8 and a double hybridization signal on the aberrant chromosome. The pattern produced by this double signal is suggestive of an inversion duplication chromosome. These studies directly confirm both the origin of the extra chromosomal material and that the duplication chromosome has undergone deletion."""@en ; edm:aggregatedCHO "https://circle.library.ubc.ca/rest/handle/2429/3323?expand=metadata"@en ; dcterms:extent "1723827 bytes"@en ; dc:format "application/pdf"@en ; skos:note "CHARACTERIZATION OF AN INVERSION DUPLICATIONOF HUMAN CHROMOSOME 8BY FLUORESCENT in situ HYBRIDIZATIONbyKAREN GWEN HENDERSONB.Sc. The University of British Columbia 1989A THESIS SUBMIIThD IN PARTIAL FULFILMENT OFTHE REQUIREMENTS FOR THE DEGREE OFMASTER OF SCIENCEinTHE FACULTY OF GRADUATE STUDIES(Genetics Programme)We accept this thesis as conformingto thç.-çqujrd.. tar1ardTHE UNIVERSITY OF BRITISH COLUMBIAOctober 1992© Karen Gwen Henderson 1992IIn presenting this thesis in partial fulfilment of the requirements for an advanceddegree at the University of British Columbia, I agree that the Library shall make itfreely available for reference and study. I further agree that permission for extensivecopying of this thesis for scholarly purposes may be granted by the head of mydepartment or by his or her representatives. It is understood that copying orpublication of this thesis for financial gain shall not be allowed without my writtenpermission.Department of 1dioQ, i7i2S’The University of British ColumbiaVancouver, CanadaDate CfDE-6 (2/88)11ABSTRACTi. ae novo cnromosomai aDerratlofl in a temale witn severe mentai retaraationand dysmorphic features has been characterized cytogenetically (Dill et al.1987). The patient’s karyotype was described as 46,XX,inv dup(8)(p12-+23.1).Previous Southern blot dosage analysis of the patient’ s DNA with a probefrom the D8S7 locus, which maps to 8p23-+8pter (Wood et al. 1986),demonstrated that the patient was monosomic for this locus. This dosageabnormality was interpreted as a consequence of the chromosomalrearrangement, suggesting that the aberrant chromosome was a duplicationdeficiency chromosome. We have reinvestigated this patient using fluorescentin situ hybridization using cosmids from a flow sorted chromosome 8 library aswell as an 8p painting probe mixture generated by Mu element mediatedPCR. Both the normal and the inversion duplication chromosome p arms areuniformly labelled by the 8p painting probe mixture. Hybridization of a cosmidfrom the D8S7 locus results in a hybridization signal on the normalchromosome 8 and a complete lack of signal on the inversion duplicationchromosome 8. Hybridization of a cosmid from the D8S 133 locus, localized to8p21-*8cen using a hybrid cell panel (Wagner et al. 1991), provides a singlehybridization signal on the normal chromosome 8 and a double hybridizationsignal on the aberrant chromosome. The pattern produced by this doublesignal is suggestive of an inversion duplication chromosome. These studiesdirectly confirm both the origin of the extra chromosomal material and thatthe duplication chromosome has undergone deletion.111i t I II I Ill I I II’ I R I’ I 4ABSTRACT. iiTABLE OF CONTENTS iiiTABLE OF FIGURES viACKNOWLEDGEMENTS viiiChapter IINTRODUCTION 11.1 In situ hybridization; History of development 11.2 Fluorescent in situ hybridization 41.3 Hybridization probes 61.3.1 Repeat sequence probes 71.3.1.1 Chromosome specific repeats 71.3.1.2 Repeat sequences found throughout thehuman genome 81.3.2 Whole chromosome and chromosome segmentpainting probes 121.3.2.1 Alu element mediated PCR - Somatic cellhybrid DNA as template 141.3.3 Locus specific probes 151.3.3.1 Cosmid probes 151.4 In situ hybridization - Applications for the analysis of cytogeneticaberrations 161.5 Project description 181.6 Thesis objectives 22ivI 1I’ IIMATERIALS AND METhODS.242.1 Established cell line 242.2 Harvest of patient’ s chromosomes 242.3. Slide preparation and storage 262.4. Cosmid DNA 262.4.1 Chromosome 8 cosmid library 262.4.2 Cosmid DNA isolation 282.4.3 Restriction enzyme digestion of cosmid DNA 292.4.4 Agarose gel electrophoresis 302.4.5 Southern transfer 302.4.6 Oligolabelling of total human DNA 312.4.7 Probe purification, spun-column chromatography 322.4.8 Blot hybridization with total human DNA 332.4.9 Washing blots hybridized with total human DNA 332.4.10 Autoradiography 342.5 Mu element mediated polymerase chain reaction 342.5.1 PCR reaction conditions 352.5.2 Purification of Alu PCR product 352.5.3 Agarose gel electrophoresis 362.6 Biotinylated probe preparation 372.6.1 Probe labelling and storage 372.6.2 Hybridization probe pre-annealing conditions 382.6.2.1 Cosmid probes 382.6.2.2 Alu PCR products 392.6.3 Hybridization probe mixture preparation 392.7 In situ hybridization 402.7.1 Hybridization conditions 402.7.2 Post-hybridization conditions 432.8 Immunocytochemical detection 432.9 Microscopy 462.9.1 Epifluorescence microscope 462.9.2 Confocal microscope 462.10 Image storage and photography 47VCHapter 111RESULTS.483.1 Cosmid DNA re-annealing conditions 483.2 Mu element mediated PCR 513.3 In situ hybridization banding (ISHB) of patient’ schromosomes 543.4 In situ hybridization 563.5 Cosmid 24E10 localization 603.6 Microscopy 62Chapter IVDISCUSSION AND CONCLUSIONS 644.1 Future studies 76REFERENCES 79vi1ALILI± O1 F1(iUKISFigure 1 A schematic representation of Mu element mediated PCR. . . 11Figure 2 Partial G-banded karyotype of patient’ s chromosomes 20Figure 3 Ideogram of human chromosome 8 indicating human content ofhybrid cell line 706B6-C117-S12 and localization of loci D8S7and D8S133 23Figure 4 A schematic representation of cosmid vector sCos-1 27Figure 5 An overview of fluorescent in situ hybridization procedures . .42Figure 6 Immunocytochemical detection of hybridization signals 45Figure 7 Agarose gel electrophoresis of an EcoRI restriction digestion ofcosmids 11E1 and 24E10 49Figure 8 Repetitive DNA content of cosmids 11E1 and 24E10 50Figure 9 Mu element mediated PCR “fingerprints” 53Figure 10 In situ hybridization banding of patient ‘s chromosomes 55Figure 11 Painting of 8p using Alu-PCR products from a somatic cellhybrid 57Figure 12 In situ hybridization of cosmid 11E1 58Figure 13 In situ hybridization of cosmid 24E10 59Figure 14 Localization of cosmid 24E10 61Figure 15 Ideogram of normal chromosome 8 and inversion duplicationchromosome 8 indicating sites of hybridization produced with theAlu-PCR painting probe and cosmids 11E1 and 24E10 67Figure 16 A proposed mechanism for the generation of an inversionduplication chromosome (Taylor et al. 1977) 71viir igure I I A proposea mecnamsm tor tfle generation ot an mversionduplication chromosome (Weleber et al. 1976 and Dill et al.1987) 74Figure 18 A proposed mechanism for the generation of an inversionduplication chromosome (Gorinati et al. 1991 and Mitchell etal.1991) 75viiiACKNOWLEDGEMENTSI would like to thank my research supervisors Drs. Stephen Wood and FredDill for their support, guidance and most especially for their patience. Thanksto my advisory committee Drs. Jan Friedman, Robert McMaster, and DavidHoim for their advice and critical reading of this thesis. Many thanks to myco-workers Heather Mitchell, Mike Schertzer, Craig Kreklywich, and LynnBernard for their friendly support and companionship. Special thanks to Dr.Vindhya Amarasinghe and Palitha Dharmawardhana for helping with confocalmicroscope image production. Their kindness and tireless help was muchappreciated.I would also like to thank my mother and father, Betty and Ron Henderson,my sister Gayle, my friends Lorne, Meriza, Nancy, Trev, Dave and my coworkers at Camgara Dental Group for their support. Finally, many thanks toCohn Savage for his confidence and endless help.1CHAPTER IINTRODUCTIONRecent developments in molecular cytogenetics have provided researchers withnew resources which can be utilized in the investigation of chromosomalaberrations. In particular, the development of techniques for fluorescent in situhybridization together with the preparation of chromosome specific and locusspecific hybridization probes (Pinkel et al. 1988; Lichter et al.1988a;Landegent et al. 1987) have provided cytogeneticists with reagents that may beused to directly characterize the structure of aberrant chromosomes. Thesetechniques are valuable in the analysis of cytogenetic rearrangementsparticularly those involving complex rearrangements, extra chromosomalmaterial or submicroscopic deletions.1.1 In situ hybridization; History of developmentIn situ hybridization procedures, which directly combine molecularhybridization and cytological material, have progressed from a laborious andtime consuming approach for the detection of abundant nucleic acid sequenceswith low resolution to an approach that allows fast, highly precise, andsensitive localization of as little as one molecule per cell (McNeil et al. 1991).Introduction 2The concept of applying molecular hybridization directly to cytologicalmaterial was pioneered by Gall and Pardue (1969) and John et al. (1969).This early technology utilized probes labelled with radioisotopes andautoradiography for the detection of abundant sequences such as thelocalization of DNA sequence in amplified polytene chromosomes (Pardue etal. 1970) or highly represented sequences on metaphase chromosomes (Evanset al. 1974). By 1981, the techniques were sufficiently refined to allowdetection of single sequences on metaphase chromosomes using 1I and 3Hlabelled probes and autoradiography (Gerhard et al. 1981; Harper et al. 1981).Although used to localize many sequences on to metaphase chromosomes thistechnique has its limitations. Isotopic in situ hybridization is very timeconsuming, often requiring several weeks for sufficient radioisotopicdisintegration to produce adequate autoradiograghic exposures. Spatialresolution is limited due to the scattering of grains that occurs on theemulsion, therefore sequence localization is not directly determined within agiven cell but requires statistical analysis of grain distribution over manychromosomes. In addition, the use of large insert probes containing repetitivesequences results in prohibitively high background signal, consequently theisolation of unique sequence is a necessity.Introduction 3In order to overcome some of these problems investigators have developeddetection techniques which utilize fluorescent or enzymatic reporter moleculesto detect non-isotopically labelled probes. Initially, detection was limited tohighly represented sequences such as satellite DNA (Manuelidis et al. 1982),amplified sequences on polytene chromosomes (Wu and Davidson 1981;Langer-Safer et al. 1982), and gene clusters for rRNA’s (van Prooijen-Knegtet al. 1982). Several improvements have been introduced which enhance thehybridization efficiency of probe molecules for target sequence withoutsubstantial non-specific adherence to biological material. These improvementsinclude, an optimization of hybridization times, probe concentrations, andprobe fragment size, as well as technical variables such as slide storage andpreparation prior to hybridization, and all result in an increase in signal tonoise ratio. Labelling and detection systems with increasing sensitivity havebeen developed such as digoxygenin-labelled nucleotides detected byantibodies carrying fluorescent or enzymatic tags (Boehringer Mamtheim).These new systems not only provide improved sensitivity of probe detection,they also provided alternate labelling and detection methods useful insimultaneous multiple probe detection experiments. In addition non-isotopic insitu hybridization can be used to resolve probe molecules separated by greaterthan lMbp on metaphase chromosomes (Trask et al. 1991b) and probemolecules separated by as little as 5Okbp on interphase nuclei (Trask et al.Introduction 41991b) with more recent studies using sperm pronuclei for very high resolutionmapping being explored. These improvements have resulted in an increasingnumber of studies demonstrating highly specific identification of unique DNAsequences in mammalian genomes, with the mapping of DNA sequences assmall as 2kb being reported (Viegas-Pequinot et al. 1989).12 Fluorescent in situ hybridizationVarious methods are available for the labelling of nucleic acidsnon-isotopically. These methods include the enzymatic incorporation ofnucleotides modified with reporter molecules such as biotin, digoxigenin,dinitrophenol (DNP), or halogenated nucleotides or the chemical modificationof DNA molecules to attach acetylaminofluorene (AAF), mercury or sulfonate.Alternatively, probe molecules can be directly conjugated with fluorescentmolecules (Bauman et al. 1980) allowing the direct detection of labelled probemolecules. This technique is now being explored for hybridization signaldetection with decreased background signal as well as for use in multicolourlabelling experiments. Labelled probe molecules between 200 and 400 bp inlength are ideal for penetration into cytological material and for networking ofprobe molecules. Probe molecules of this size are produced either directlythrough nick translation labelling or by sonication after labelling. TheIntroduction 5cytological material is prepared, with care taken to preserve target sequencesin an accessible state and the labelled DNA molecules are then hybridized tothe cytological material. Hybridized probe molecules can be visualized in anumber of ways; via fluorochromes and fluorescence microscopy,chemiluminescence detected by an emulsion overlay or by colour precipitatesgenerated by enzymatic assays or the use of colloidal gold for visualization byphase contrast or electron microscopy. Fluorochrome conjugated avidin,streptavidin, or anti-biotin antibodies are used to detect probes labelled withbiotin. Digoxigenin, DNP, AAF or sulfonate are labelled with specificantibodies which are detected with fluorescence labelled anti-immunoglobulins.A variety of fluorochromes are available with emission spectra ranging fromblue upon UV excitation to infrared after red light excitation. The mostcommon fluorochromes being used are fluorescein isothiocyanate (FITC)which is excited with blue (490nm) light and emits green (525nm) light, Texasred which is excited with yellow (590nm) light and emits orange (615nm) lightand rhodamine isothiocyanate (TRITC) which is excited with green (540nm)light and emits yellow (550nm) light. Brighter signals can be obtained byantibody amplification for biotinylated probes. Signal amplification is achievedby the successive layering of components. For example, a biotinylated probe isdetected with streptavidin conjugated with FITC. The fluorochromes signal isIntroduction 6then increased by layering biotinylated anti-streptavidin antibody andre-conjugating with streptavidin FITC.Recent developments in optical instrumentation have also contributed to theincreased sensitivity and application of fluorescent in situ hybridization. Inparticular, the improved signal detection capabilities of optical instruments,such as CCD (charged coupled device) cameras, filter sets which allow thesimultaneous visualization of two fluorochromes (such as Texas red andFITC), the development of confocal microscopes for three dimensionalmicroscopy and computer imaging technology are all being used to supplementconventional epifluorescence microscopy.1.3 Hybridization probesThe usefulness of fluorescent in situ hybridization depends primarily on theavailability of probes or probe sets which hybridize specifically to regions ofgenetic or cytogenetic interest. In general there are three probe typesavailable; the repeat sequence probes, chromosome or segment specific probesand locus specific probes. Repeat sequence probes can be classified into twogroups; those for repeat sequences found primarily on one chromosome typesuch as the alpha satellite repeats and those repeat sequences foundIntroduction 7throughout the genome such as Alu or Li sequences. The chromosome orsegment specific probes, sometimes called painting probes, are probe setswhich can be used to highlight whole chromosomes or segments ofchromosomes. Finally, locus specific probes are those used to label uniquesequences in the genome and are cloned DNA in plasmid, phage, cosmid oryeast artificial chromosome (YAC) vectors.1.3.1 Repeat sequence probes1.3.1.1 Chromosome specific repeatsHuman chromosomes contain DNA sequences in the centromeric regionswhich are tandemly repeated several hundred to several thousand times (Wayeet al. 1987). These sequences, belonging primarily to the alpha-satellite or thesatellite-Ill families (Fowler et al. 1989), have been isolated and cloned formost human chromosomes. There is sufficient variation in sections of thetandem repeats to allow for chromosome specific hybridization, producing anintense and compact signal near centromeres or in heterochromatic regions ofspecific chromosomes.Introduction 81.3.1.2 Repeat sequences found throughout the human genomeSeveral families of interspersed repeat sequences are found in high copynumber in mammalian DNA. Two notable examples are the Mu sequencesand the Li sequences. The Mu sequence family is the major member of theSINES (short interspersed repeat sequences) families of repeat elements. It isapproximately 300 bp long, and is found in approximately 300,000-900,000copies in the human genome (Britten et al. 1988). The Li sequence family isthe major member of the LINES (long interspersed repeat sequences) familiesof repeat elements. It is approximately 6.4 kb long, and is found inapproximately 4,000-100,000 copies in the human genome (Grimaldi et al.1984). A non-random distribution of these sequences has been demonstratedcytologically (Korenberg and Rykowski 1988; Chen and Manuelidis 1989). Murepeats have been shown to cluster preferentially in Giemsa light bandswhereas Li repeats cluster preferentially in Giemsa dark bands. Hybridizationof these interspersed repetitive elements would therefore produce G or Rbanding patterns on metaphase chromosomes and would be useful for thegeneration of an in situ hybridization banding (ISHB) profile. Both cloned Murepeats and PCR products produced using an Alu primer have been reportedto produce banding patterns (Lichter et al. 1990; Baldini et al. 1991). The Lisequence of mouse has been used to generate high quality Giemsa likebanding in mouse chromosomes (Boyle et al. 1990), but to date neither LiIntroduction 9clones or Li PCR products have generated true G-banding patterns inhumans.Mu elementsThe members of the Mu DNA repeat family are found concentratedpredominantly in G negative or R bands. Constitutive heterochromatic regionsof human chromosomes as well as the heterochromatic regions below thecentromeres of chromosomes 1, 3, 6, 9, 16, and the long arm of the Ychromosome contain few if any Mu elements (Manuelidis and Ward 1984).R-banding produced by fluorescent in situ hybridization with a cloned Muelement as the probe has been used for chromosome identification (Lichter etal. i990a), but the quality of banding proved to be variable. The recentdevelopment of Mu element primed amplification of human DNA using thepolymerase chain reaction allows the amplification of sequences between Muelements (Nelson et al.1989). When the resulting product is hybridized tohuman chromosomes, a well defined and reproducible R-banding patternresults (Baldini et al. 1991).Mu element mediated PCR - Total human DNA as templateMu PCR involves the use of primers corresponding to the consensus sequenceof the Mu family of repetitive elements to amplify the DNA found betweenIntroduction 10two adjacent Alu elements (Nelson et al. 1989). A single oligonucleotideprimer homologous to the human Mu repeat element anneals to denaturedtemplate DNA and amplifies sequences found between two oppositely orientedMu elements which are at a distance amenable to PCR amplffication.Successive cycles of denaturing of template DNA, annealing of Mu primers,and extension or synthesis of inter-Mu DNA amplifies DNA between annealedprimers (Figure 1). Since Mu sequences are concentrated in Giemsa lightstaining regions of human chromosomes, the Mu PCR product will beenriched in sequences from these regions.Introduction 11ALU ELEMENT MEDIATED PCR-Alu elements inopposite orientationsExponentialAmplificationFigure 1 Alu element mediated PCR. A single oligonucleotide primer (A1S)homologous to the extreme 3’ end of the human Mu repeat element is usedto amplify DNA found between two oppositely oriented and appropriatelyspaced Mu elements.IMS p.imar‘13’3’prm.r!!Al S!!Introduction 12In situ hybridization banding (ISHB)Hybridization of Alu-PCR products generated using total human DNA astemplate produces a distinct and reproducible R-banding pattern that is similarto a conventional R-banding pattern. Some variation from standard R-bandingis observed specifically at the constitutive heterochromatic regions ofchromosomes which contain few, if any, Mu repeats and therefore produce nofluorescent signal upon hybridization of Alu-PCR product. In addition, thepericentromeric heterochromatic regions of chromosomes 1, 3, 9, 16 and onthe long arm of the Y contain few Mu repeats so there is a complete oralmost complete lack of fluorescence in these regions (Baldini et al. 1991).This high quality chromosomal banding can be used to produce a bandedkaryotype and would also be useful in double labelling experiments for thesimultaneous production of a banded karyotype and the localization of probesto specific chromosome bands.1.3.2 Whole chromosome and chromosome segment painting probesCollections of DNA sequences derived from a single human chromosome orchromosomal segment can be used to highlight a chromosome or region byhybridization to metaphase or interphase chromosomes. Such DNA collectionscan be obtained from chromosome specific recombinant DNA librariesIntroduction 13(Lichter et al. 1988a; Cremer et a!. 1988; Pinkel et al. 1988), somatic cellhybrids containing the desired chromosome or chromosome segment as thesole human material (Kievits et al. 1990), or suspensions of wholechromosomes, chromosome subregions or aberrant chromosomes purified byflow sorting (Ferguson-Smith 1991). These probe mixtures contain repetitivesequences common throughout the genome, and in order to achieve thedesired staining contrast, pre-hybridization with unlabelled competitor DNA isrequired. The sensitivity and accessibility of these painting probe sets issomewhat limited. The availability of recombinant DNA libraries restricted tochromosomes and segments of interest is limited. The chromosome or segmentof interest present in a hybrid cell line represents only a small fraction of thetotal DNA content, this results in decreased sensitivity as well as a need tooptimize pre-annealing conditions to eliminate cross-hybridization of hamstersequences which have homology to human sequence. To overcome theselimitations, PCR amplification using A!u directed oligonucleotide primers canbe used to generate probes from somatic cell hybrid DNA containing thechromosome or segment of interest. This will produce a complex probe sethighly enriched in the human component of the cell line. This mixture canthen be hybridized to human metaphase spreads resulting in the painting ofthe chromosome or region of interest.Introduction 141.3.2.1 Mu element mediated PCR- Somatic cell hybrid DNA as templateAlu PCR utilizes oligonucleotide primers, which correspond to the consensussequence of the Mu family of repetitive elements, to amplify human DNAfound between two adjacent Mu elements (Nelson et al. 1989). A primerhomologous to the human Mu repeat element provides the basis forpreferential synthesis of human DNA fragments from a human/rodent somaticcell hybrid DNA template. DNA can be synthesized between Alu elementswhich are in opposite orientation and within a distance appropriate for PCRamplification. Although rodent DNA contains sequences homologous tohuman Mu elements, these repeat elements are less well conserved betweenspecies than they are within the human species (Jelinek and Schmid 1982)allowing for preferential amplification of the human sequences from hybridDNA. An Mu PCR system which uses a primer homologous to the extreme 3’end of the repeat element, and designed in such a way that DNA synthesisoccurs away from the Mu element, will generate DNA which is almostcompletely free of Mu sequence with the exception of the primer sequenceitself. During amplification repetitive non-Mu sequences will be amplified soinitial pre-annealing to suppress this repetitive content is required to producehighly specific painting of chromosomes or regions of interest.Introduction 151.3.3 Locus specific probesOnce loci have been identified, they can be studied using fluorescent in situhybridization with probes for the regions of interest. Plasmid probes containingas little as 2 kb of target sequence have been localized using fluorescent in situhybridization (Viegas-Pequignot et al. 1989). The efficiency of hybridizationsignal detection with these small probes is approximately 50% with theefficiency of detection increasing with increased probe size (Trask 1991a).DNA sequences cloned into large insert phage (Pinkel et al. 1988), cosmids(Lichter et al. 1990b) or yeast artificial chromosomes (Montanaro et al. 1991)have been used successfully as locus specific hybridization probes.1.3.3.1 Cosmid probesHybridization of large unique DNA segments to metaphase chromosomes ispossible with the initial suppression of non-specific signal. Non-specific signals,due to the presence of repetitive sequences in cloned DNA segments, may besuppressed using an excess of unlabelled appropriate competitor DNA. Thisprocess leads to the preferential reassociation of repetitive sequences andpermits the hybridization of unique sequences to the chromosomes (Lichter etal. 1988a). The efficiency of labelling unique sequences using these large insertprobes is greater than 90% under suppression conditions (Trask et al., 1989b;Introduction 16Lichter et al., 1990b; Ferguson-Smith 1991). Both chromatids are usuallylabelled giving twin hybridization spots within the chromatin. This provides aninternal positive control and in general only a small number of metaphasesneed to be examined and statistical analysis is not necessary (Ferguson-Smith1991).1.4 In situ hybridization - Applications for the analysis of cytogeneticaberrationsThe speed and resolution of in situ hybridization as well as the increasingaccessibility of a variety of probes has made it feasible to apply this techniquefor the characterization of cytogenetic aberrations. The usefulness offluorescent in situ hybridization for the detection of chromosomal aneuploidyhas been demonstrated in both interphase (Cremer et al. 1986) and metaphase(Lichter et a!. 1988b) chromosomes. The characterization of rearrangementssuch as inversions and translocations have been achieved using fluorescent insitu hybridization and chromosome specific probes. The BCR-ABL fusionevent associated with chronic myelogenous leukaemia has been detected usingfluorescent in situ hybridization with probes from portions of the bcr and ablgenes (Tkachuk et al. 1990). An inversion chromosome characteristic of typeIntroduction 17M4 acute nonlymphocytic leukaemia has been visualized using fluorescent insitu hybridization with two strategically located cosmid probes (Dauwerse et al.1990).Complex rearrangements are very difficult to interpret cytogenetically. Boththe nature of the rearrangement itself and the origin of the extra chromosomalmaterial is difficult to establish. The use of chromosome specific or segmentspecific hybridization probes allows for quick and definitive identification ofthe origin of extra chromosomal material. A 12p painting probe mixture hasbeen used to demonstrate the genuine iso-12p character of the standardmarker chromosome of testicular germ cell tumours (Suijkerbuijk et al. 1991).Fluorescent in situ hybridization using a locus specific probe has been used todemonstrate the amplification of the dihydrofolate reductase gene in Chinesehamster ovary cells grown for an extended period in methotrexate (Trask et al.1989a).The identification of small deletions is one of the most challenging tasks ofcytogenetic analysis. Submicroscopic deletions involving a few kilobases ormore of DNA are readily apparent by fluorescent in situ hybridization. Thehigh efficiency of nonisotopic in situ hybridization with cloned genomic DNAfragments permits such an analysis on both metaphase and interphaseIntroduction 18chromosomes. The usefulness of this approach has been demonstrated for thedetection of a deletion of the ankyrin gene resulting in a subtype of hereditaryspherocytosis (Lux et a!. 1990), detection of submicroscopic deletions in threepatients with Miller-Dieker syndrome (Kuwano et al. 1991), deletion detectionin Angelman/Prader-Willi syndrome patients without visible rearrangements(Wagstaff et al. 1991) as well as for the demonstration of the carrier status ofwomen with deletions in the dystrophin gene (Ried et al. 1990).1.5 Project description8p inversion duplication patientG.S. is a 32 year old profoundly retarded female who resides in an institution.She was born to a 20 year old mother and a 23 year old father. Her birthweight was 3 100g. Developmental milestones were markedly delayed and atage three years and four months she was noted to be grossly retarded. She hasprofound scoliosis extending from T8 to LA and dysmorphic features.Chromosome analysis of the patient and her parents was carried out oncultured lymphocytes using conventional G-banding. The patient ‘5 karyotypeIntroduction 19revealed additional bands on 8p. The karyotypes of both parents were normal.Subsequent prometaphase analysis of the abnormal chromosome 8 suggestedthat the extra bands represented an inverted duplication involving bands8p12-23.1 (Dill et al. 1987). In addition, marked banding symmetry around apoint distal to band 8p22 was noted. The patient’s karyotype was assigned as46,XX,inv dup(8)(p12-i23.1) (Figure 2).Introduction 20KFigure 2 Partial G-banded karvotvpe of patient ‘ s chromosomes. This figureshows two cells, in each cell the normal chromosomes 8 are on the left and theinversion duplication chromosomes 8 are on the right (Dill et al. 1987).Introduction 21Southern blot dosage studies with a probe for the D8S7 locus, which has beenlocalized to 8p23-*8pter (Wood et al. 1986), demonstrated that the patient wasmonosoniic for this marker. This dosage abnormality was interpreted to be aconsequence of the chromosome abnormality, indicating that the aberrantchromosome was a duplication deficiency chromosome (Dill et al. 1987). Thisdosage result was completely unanticipated as there was no evidence ofdeficiency from the karyotype.Among the inversion duplication 8p patients reported at the time (Weleber etal. 1976; Rethoré et al. 1977; Taylor et al. 1977; Mattei et al. 1980; Jensen etal. 1982; Fryns et al. 1985; and Kleczkowska et al. 1987), five were reported tohave a distal deficiency (Weleber et al. 1976 Rethorê et al. 1977 Mattei et al.1980 and Jensen et al. 1982). Of the more recent cases reported (Poloni et al.1981a, 1981b; Nevin et al. 1990; Mitchell et al. 1991 and Gorinati et al. 1991),an inversion duplication (8)(p2l.l-22.l) patient has been reported where highresolution banding led to the conclusion that 8p23.2 was deleted (Gorinati etal. 1991), although no molecular studies were performed. An additionalinversion duplication (8)(p2l.l.+23.l) patient has been reported (Mitchell et al.1991) where DNA dosage studies were consistent with a distal deficiency.Introduction 22Consequently, a distal deficiency may be a common feature of inversionduplication chromosomes.1.6 Thesis objectivesThe purpose of this project was to reinvestigate and characterize patientG.S. ‘s inversion duplication chromosome 8 using fluorescent in situhybridization. The source of extra chromosomal material was determined usingan 8p chromosome painting probe generated by Alu element mediated PCR,using DNA from the hamster-human hybrid cell line 706B6-Cl17-S12 (Woodet al. 1992) (Figure 3) as template. In addition two cosmids 11E1 (D8S7 locus)and 24E10 (D8S133 locus) (Figure 3), isolated from the LAO8NCO1 flowsorted cosmid library (Wood et al. 1992) and carefully chosen to representeither a suspected deletion (11E1) or duplication (24E10) region of theaberrant chromosome, were hybridized to the patient’ s chromosomes in aneffort to provide definitive evidence of both a duplication and a deletion ofchromosomal material.HUMAN CHROMOSOME 8Introduction 23Figure 3 Ideogram of human chromosome 8 indicating human content ofhybrid cell line 706B6-C117-S12 and localization of loci D8S7 and D8S133.Hybrid cell fln•706a6—c117—S1223.3 (- 123.208S723.1 [ j J2221.321.221.11208S13311.2111.2211.23_________121321.121.221.322.122.222.32324.124.224.324Chapter IIMATERIALS AND METHODS2.1 Established cell lineAn Epstein Bar virus transformed lymphoblastoid cell line, established frompatient G.S. and carrying a de novo inversion duplication (8)(p12-+p23.l), wasmaintained in continuous suspension culture. The cells were grown in RPM!1640 medium (Gibco BRL) supplemented with 15% foetal bovine serum(Bockneck Laboratories). Media was occasionally supplemented withadditional L-glutamine (Gibco BRL) at a concentration of 146mg/500m1 whenthe cells were growing slowly and (or) RPM! 1640 media was several weeksold. The cultures were split biweekly to a concentration of 5x10 cells permilliliter to maintain optimum growth.2.2 Harvest of patient’ s chromosomesChromosomes were prepared according to standard protocols (Verma andBabu 1989) with minor modifications. Approximately thirty six hours after thelymphoblastoid culture had been split to a concentration of 5x10 cells permilliliter, 8m1 aliquots of cells were removed and placed into 15m1 conicalculture tubes. This suspension was then pipetted several times, using a lOmipipette, to break-up the larger cell clumps. To synchronize the cell growth,Materials and Methods 255-fluoro-2 ‘ -deoxyuridine (FUDR) (Sigma) at a concentration of 0.009 g per8m1 culture was added, the cultures were mixed well and the time noted. Thecells were then incubated at 37°C for 14-18 hours. After this incubation, thecells were arrested in metaphase by the addition of colcemid (Gibco) at aconcentration of 1J.Lg per 8m1 culture. The cultures were then incubated at37°C for 13 minutes and were then centrifuged at 1000rpm for 10 minutes. Thesupernatant was discarded and the cell pellet was re-suspended in about lOmiof a hypotonic (0.075M) potassium chloride solution, and incubated once againat 37°C for 18 minutes. After incubation, two drops of cold (-20°C) 3:1 fix (3:1absolute methanol:glacial acetic acid) were added to each tube, the tubes weremixed by inversion and the cells were then centrifuged at 1000rpm for 10minutes. The supernatant was discarded and the cell pellet was re-suspendedin lOmi of 3:1 fix and placed at 4°C for a minimum of 20 minutes. Thiscompletely stops all cell processes as well as the action of the colcemid. Thecells were then washed in 3:1 fix twice or until the cell pellet was free ofdebris. At this point, either metaphase spreads were prepared or the cell pelletwas stored at 4°C until needed.Materials and Methods 262.3. Slide preparation and storageOnce a clean pellet of cells had been prepared, the cells were diluted inenough 3:1 fix to make a milky suspension. Pre-cleaned slides were handwashed in a mild detergent and rinsed profusely in dH2O. The cell suspensionwas added dropwise to the upper right corner of each slide and the slide wasgently tipped to allow the suspension to flow across and down the slide. In thisway a reasonably dense, uniform and well spread selection of metaphases andnuclei were distributed across the entire slide. Slides were allowed to air dryand were used for in situ hybridization within 24 hours. In some cases air driedslides were desiccated and stored at -70°C for future use (Jauch et al. 1990).2.4. Cosmid DNA2.4.1 Chromosome 8 cosmid libraryThe source of cosmid DNA was the chromosome 8 flow sorted libraryLAO8NCO1. This library was constructed in the cosmid vector Scos-1 (Figure4), with inserts of partially digested Sau3AI chromosome 8 DNA cloned intoBamHI cleaved Scos-1 vector cloning sites. The average cosmid size in thislibrary is 43.4kb. Since the Scos-1 vector is 6.9kb the average insert size istherefore 36.5kb (Wood et al. 1992).Materials and Methods 27OnAmpKan •:•sO os 16900 bp:&,‘‘ CsCos£co RI 17 CO RINell BomMi NotiI I I I IFigure 4 A schematic representation of cosmid vector Scos-1. The basicfeatures include the ColE 1 origin of replication (On), the ampicilhin (Amp)and kanamycin (Kan) resistance genes, two cos packaging signals (Cos) from )vector Charon 4A., and bacteriophage T3 and 17 promoter sequences flankingthe cloning site (C.S.).Materials and Methods 282.4.2 Cosmid DNA isolationCosmid mini-preps were prepared by alkaline lysis (Birnboim and Doly 1979;Ish-Horowicz and Burke 1981). E. coli containing the appropriate recombinantcosmid clone were streaked on agar plates containing kanamycin or ampicillinand grown overnight at 37°C. A single bacterial colony was transferred to 5m1of L-broth (5g yeast extract, lOg tryptone, 5g NaC1, ig dextrose and dH2O to 1liter) containing 5jg/m1 kanamycin, grown overnight at 37°C and pelleted. Thepellet was suspended in 2OOil of solution I (50mM glucose, 25mM Tris Cl pH8.0, 10mM ethylenediaminetetraacetate (EDTA) pH8.0) containing 4mg/ml oflysozyme (Sigma) and incubated for 5 minutes at 20°C. To this, 4001il offreshly prepared solution II (0.2N NaOH, 1% sodium dodecyl sulfate) wasadded and the mixture was then incubated for 5 minutes on ice. Finally, 300i.tlof solution III (3M potassium acetate, 2M acetic acid) was added and then themixture was incubated for 5 minutes on ice. The mixture was then spun in amicrofuge at 14,000g for 10 minutes at 4°C. The clear supernatant wasrecovered and extracted with 600.d of phenol:chloroform:isoamyl alcoholsolution (25:24:1). The top aqueous layer was transferred to a new microfugetube to which one half volume of 7.5M ammonium acetate and two volumes of95% ethanol had been added and the double stranded DNA was allowed toMaterials and Methods 29precipitate at -20°C for thirty minutes to two hours (Maxam and Gilbert 1980).The mixture was then centrifuged at 14,000g for 10 minutes at 4°C, thesupernatant was removed and the tube inverted to allow all fluid to drain.The DNA pellet was then rinsed with lml of 70% ethanol, centrifuged anddried in a vacuum desiccator. Finally, the pellet was redissolved in 20.Ll TE(10mM Tris, 1mM EDTA) pH 8.0 containing 2.Ll of DNase-free pancreaticRNase (20kg/mi) (Sigma) and incubated at 37°C for one hour. The preparedcosmid DNA was stored at -20°C.2.4.3 Restriction enzyme digestion of cosmid DNAApproximately 1j.g of cosmid DNA was combined with 2l of lox BSA(bovine serum albumin fraction V 1mg/mi) (Sigma), 2d of lOX React 3 buffer(BRL) and dH2O to a volume of 201il. One to two units of restriction enzymeEcoRI (BRL) was added and the reaction mixture was incubated at 37°C forone and a half hours. The reaction was stopped by the addition of 0.5MEDTA pH 8.0 to a final concentration of 10mM. To this, 1/10 volume ofloading buffer (0.25% xylene cyanol, 0.25% bromophenol blue, 40% sucroseW/V in water) was added and the mixture loaded onto an agarose gel.Materials and Methods 302.4.4 Agarose gel electrophoresisRestriction digests of cosmid DNA were combined with loading buffer andloaded on to 0.8% agarose (Pharmacia) gels. The DNA fragments were sizeseparated by electophoresis in 1X Tris-borate buffer (0.090M Tris-borate,0.002M EDTA) at 30 volts for 16 to 18 hours. Ethidium bromide was presentin the agarose gel at a concentration of 0.lmg/lOOmi TBE. Molecular weightswere determined using Hind!!! Saclil digested lambda DNA as the molecularweight standard. The gel was photographed under ultraviolet light.2.4.5 Southern transferOnce the DNA had been digested, size separated by electrophoresis throughan agarose gel, and a photograph of the ethidium bromide stained gel hadbeen taken, the gel was placed in a Pyrex dish and treated as follows: a 25minute wash in 0.25M HC1 to de-purinate the DNA, a 30-60 minute wash in1.5M NaC1/0.5M NaOH to denature the DNA followed by neutralization in1M Tris, 1.5M NaC1 for 30 minutes. A Southern blot was then set-up with theorder of materials from bottom to top as follows: Pyrex dish filled with lOXSSC buffer (87.6g NaC1, 44.lg sodium citrate and dH2O to 1 liter), glass plate,3MM Whatman paper with ends soaking in lox SSC, agarose gel, Hybond-N(Amersham) membrane cut to size of gel and wetted with H20, one sheet of3MM Whatman paper cut to size of gel and wetted with water, one sheetMaterials and Methods 313MM Whatman paper cut to size of gel and dry, plastic wrap surrounding andup to gel borders, and approximately 10 cm of paper towels. The dry papertowels draw the lox SSC buffer upward by capillary action and provideunidirectional transfer of the DNA onto the membrane (Southern, 1975). Thetransfer was allowed to proceed for 16 hours, at which time the Hybond-Nmembrane was baked at 80°C for two hours to fix the DNA onto themembrane. Finally, the efficiency of DNA transfer was checked by re-stainingthe gel with ethidium bromide and viewing with an ultraviolet light.2.4.6 Oligolabelling of total human DNATotal human DNA was radiolabelled by the oligolabelling procedure ofFeinberg and Vogeistein (1983 and 1984). Human DNA (l5ng) in a volume of15j.tl was denatured by boiling for 10 minutes and placed on ice. To this 2.5ilof iox BSA (1mg/mi), 5jil of oligolabelling buffer, 2.51 [alpha 32P]-dATP(3000Ci/nimol), and one unit of Kienow polymerase (Pharmacia) was added.The reaction was incubated at room temperature overnight and stopped by theaddition of 25l stop buffer (50mM EDTA, 20mM NaC1, 0.1% SDS and500g/mi sheared salmon sperm DNA).Materials and Methods 32Oligolabelling bufferOligolabelling buffer was made using solutions A, B, and C in a ratio of 2:5:3.Solution A: imi 1.25M Tris Cl pH 8.0, 0.125M MgC12, 18il betamercaptoethanol, and 5l each of 100mM dGTP,dCTP, and dTFPSolution B: 2M HEPES pH 6.6Solution C: 90 OD U/mi randomly generated hexanucleotides.2.4.7 Probe purification, spun-column chromatographyThis method was used to separate labelled probe molecules, which passthrough the gel-filtration matrix, from lower molecular weight unincorporatednucleotides which are retained on the column. The column was made byplugging the bottom of a imi pipette tip with about 4mm of sterile sialinizedglass wool. This tip was then placed into a 1.5ml eppendorf tube of which thebottom 1cm had been cut off. The tip and eppendorf tube were then placedinto a 5m1 sterile tube. The plugged pipette tip was filled with Sephadex G-25superfine which had been equilibrated in a 1X TE:dH2O (1:5) solution. Theresin was added until the pipette tip was completely full. The column was thencentrifuged at 1600g for 2 minutes at room temperature in a swinging-bucketrotor in a bench centrifuge. The spun column was placed into a fresh sterile5m1 tube, the DNA sample was added to the column, and re-centrifuged atMaterials and Methods 331600g for 2 minutes. The purified DNA sample, which has been eluted fromthe column, was collected.2.4.8 Blot hybridization with total human DNAThe baked Hybond-N membrane was placed into a heat-sealable bag; care wastaken not to touch the membrane with fingers. About 20m1 of hybridizationsolution (6X SSC, 5X Denhardt’ s reagent, 0.5% SDS, 100g/l denaturedsheared salmon sperm DNA) and 501il of purified denatured oligolabelledprobe mixture was added. The hybridization bag was heat sealed taking care toremove all air bubbles and placed into a plastic container holding about oneinch of water. The membrane was incubated with continual shaking at 65°Covernight.2.4.9 Washing blots hybridized with total human DNAThe heat sealed bag was cut open and the probe mixture discarded. Themembrane was washed with 500 ml of 1X SSC, 0.1% SDS at roomtemperature with shaking for 15 minutes. The membrane was then transferredto 500m1 of 0.2X SSC, 0.1% SDS and washed for 45 minutes to 1 hour at65°C. The solution was then drained from the membrane, excess fluid wasblotted from the membrane using ifiter paper and the membrane was wrappedin plastic wrap.Materials and Methods 342.4.10 AutoradiographyThe wrapped membrane was taped onto a piece of Kodak XAR film, placedinto a cassette with DuPont Lightening Plus intensifying screens, and overlaidwith a piece of Kodak XRP film. The cassette was stored at -70°C andapproximately four hours later the XRP film was developed to determine theprogress of the exposure. After 16-24 hours the remaining film was developed.2.5 Mu element mediated polyinerase chain reactionAlu element mediated PCR was performed using DNA from thehamster-human hybrid cell line 706B6-C117-S12 (Wood et al. 1992) containingchromosome 8p as its only human component, hamster-human hybrid cell line706B6-C117 (Jones et al. 1981; Dalla-Favera et al. 1982) containingchromosome 8 as its only human component and total human DNA astemplate. The primer used was the A1S primer of Brooks-Wilson et al. (1990).This primer has complete homology to the extreme 3’ end of the Aluconsensus sequence of more than 40% of human Alu elements (Kariya et al.1987) with 5’ modifications for cloning (ten 5’ nucleotides comprise a Sailrecognition site plus four extra 5’ residues). The sequence of the A1S primeris 5’ TCATGTCGACGCGAGACTCCATCTCAAA3’. This primer was madeusing an applied Biosystems 380B oligonucleotide synthesizer and was purifiedby a C-18 Sep-Pak procedure.Materials and Methods 352.5.1 PCR reaction conditionsPCR reactions were done in a total volume of 50l. lOOng of somatic cellhybrid DNA or lOng of total human DNA was used as template and 1.25U ofCetus Taq polymerase. The reaction conditions were 50mMtris(hydroxymethyl) aminomethane pH 8.0; 0.05% Tween-20; 0.05% NP-40;1.8mM MgC12; 200uM each of dATP, dCTP, dGTP and dTrP; and 0.5uMA1S primer. Twenty-five cycles of a one minute denaturation at 94°C, a twominute annealing at 58°C, and a three minute extension at 72°C, with anadditional 10 second increase per cycle and a final 72°C incubation for 10minutes were performed. A Perkin-Elmer Cetus thermal cycler was used. AluPCR products were separated by size with gel electrophoresis on a 0.8%agarose gel producing PCR “fingerprints” for each of the somatic cell hybrids.The gel was stained with ethidium bromide and photographed underultraviolet light.2.5.2 Purification of Alu PCR productThe PCR reaction mixture was combined with 2001ilphenol:chloroform:isoamyl alcohol solution (25:24:1), mixed well, andcentrifuged at 14,000g for 5 minutes. The aqueous layer was recovered andcombined with 150l Seavag (24:1 chloroform:isoamyl alcohol) and centrifugedMaterials and Methods 36at 14,000g for 5 minutes. The aqueous layer was again recovered and the DNAprecipitated with cold (-20°C) 95% ethanol and centrifuged at 14,000g for 15minutes. The pellet was lyophffized and re-suspended in 10Od 5mM Tris HC1pH 8.0, 0.1mM EDTA. NaC1 was added to a concentration of 0.1M and theDNA was re-precipitated in two volumes of cold (-70°C) ethanol. The pelletwas washed in 70% ethanol, desiccated, and re-suspended in 50l TE. TheDNA was stored at -20°C.2.5.3 Agarose gel electrophoresisPurified Mu PCR products were combined with loading buffer, loaded on to0.8% agarose (Pharmacia) gels, and size separated by electrophoresis in 1XTris-borate buffer (0.090M Tris-borate, 0.002M EDTA) at 30 volts for 16 to 18hours. Ethidium bromide was present in the agarose gel at a concentration of0.lmg/lOOmi TBE. Molecular weights were determined using Hindifi Saclildigested lambda DNA as the molecular weight standard. The gel wasphotographed under ultraviolet light.Materials and Methods 372.6 Biotinylated probe preparation2.6.1 Probe labelling and storageCosmid DNA and Alu PCR DNA was biotinylated using a BioNick labellingsystem (Bethesda Research Laboratories Life Technologies, Inc.). Eachlabelling reaction was monitored by running a parallel radioactively spikedreaction and using the measure of incorporated radioisotope to estimate thelevel of biotin incorporation. The proportion of the radioactive precursorwhich had been incorporated into the desired product was measured bydifferential precipitation of the nucleic acid products with trichioroacetic acid(TCA) and ratio quantification of the specific activity of the radioactivesamples. This quantification was done by a standard trichloroacetic acid assay(Sambrook et a!. 1989). Briefly, a 11 sample of the [alpha 32P]-dATP spikedreaction mixture was removed and combined with 1OOil of stop buffer (50mMEDTA, 20mM NaCl, 0.1% SDS, and 500jg/ml sheared salmon sperm DNA).This mixture was then split into two 401il aliquots and placed into separatemicrofuge tubes. One tube was used as a measure of the total radioactivity inthe sample, to this tube 10Ol of dH2O was added. The other 4Ob’l sample wasused to determine incorporated nucleotides, to this tube 10Ol of ice cold 10%TCA (50g TCA in 227m1 dH2O) was added, mixed well and centrifuged at14,000rpm for two minutes. The supernatant, containing unincorporated [alpha32P]-dATP nucleotide was discarded and 1001 of dH2O was added to thisMaterials and Methods 38tube. Both tubes were read in a scintillation counter and an F ratio wascalculated (F = counts per minute in the TCA treated sample / counts perminute in the untreated sample). 32P can be detected in this way by Cerenkovcounting in the 32P channel of a liquid scintillation counter.Those reactions which had an F ratio of 0.25 or greater indicating 25%incorporation of [alpha322P]-dATP were considered a success and indicatedan adequate level of biotinylation in the standard reaction mixture. At thispoint the standard reaction mixture was purified to removed unincorporatedbiotin-14-dATP. Purification was done by chromatography through a columnmade with Sephadex G-25 superfine. Purified labelled probe DNA was storedat -20°C until used and is stable at this temperature for about one year.2.6.2 Hybridization probe pre-annealing conditions2.6.2.1 Cosmid probesTo determine the appropriate pre-annealing times required for each cosmid anestimate of repetitive sequence content of each cosmid was determined. Arestriction enzyme digest (EcoRI) of each cosmid was run overnight on a 0.8%agarose gel at 30 volts to ensure clear resolution of all EcoRI bands. This gelwas then stained with ethidium bromide, photographed and a Southern blotwas set-up to transfer the DNA to a Hybond-N membrane. Once the transferMaterials and Methods 39was complete, the membrane was baked for 2 hours at 80°C and probed withtotal human DNA. Hybridization and washing were done as described insections 2.4.8 and 2.4.9 and two pieces of Kodak film were exposed, one for 3hours and the other overnight. These films were then compared to thephotograph of the ethidium stained gel and an estimate of relative content ofrepetitive sequence was determined for each cosmid by observing the numberand intensity of EcoRI bands which hybridize total human DNA.2.6.2.2 Alu PCR productsAlu PCR product re-annealing times were determined empirically. Identicalprobe mixtures were prepared and allowed to partially anneal for 0, 15, 30,and 60 minutes.2.6.3 Hybridization probe mixture preparationBiotinylated cosmid DNA (6Ong) was mixed with 3jLg of unlabelled shearedhuman placental DNA while bOng of biotinylated Alu PCR product wasmixed with 3g of both unlabelled sheared human placental DNA andunlabelled sheared salmon sperm DNA. These DNA mixtures were thenlyophilized, re-suspended in 15il of formamide (pH 7.0) and denatured byheating to 70°C for 5 minutes. Probe DNA was then combined with l5J.Ll ofhybridization buffer consisting of 6jLl 50% (W/V) dextran sulfate: 31il loxMaterials and Methods 40BSA: 3l 20X SSC: 31il dH2O. Biotinylated DNA was allowed to partiallyanneal at 37°C to suppress repetitive sequences (Lichter et al. 1988a; Lengaueret al. 1990). A ten minute re-annealing time was used for cosmid 11E1, fiftyminutes for cosmid 24E10 and thirty minutes for Alu PCR DNA. The finalhybridization solutions were 50% formamide (V/V), 2X SSC (Ph 7.0), 10%(W/V) dextran sulfate, cosmid DNA 2ng/J.Ll, placental and salmon spermDNA 100ng/1 and Mu PCR DNA 3.3ng/l. The cosmids were co-hybridizedwith biotinylated alpha satellite DNA D8Z1 (Oncor) at 0.5ng/l.The Oncor alpha satellite probe was shipped in hybrisol VI, a 65% formamidebased solution. The alpha satellite probe hybridized well on its own in thissolution but upon co-hybridization, with the cosmids, better results wereobtained when the Oncor probe was lyophilized and re-suspended in 50%formamide.2.7 In situ hybridization (Figure 5)2.7.1 Hybridization conditionsPrior to hybridization, chromosome slides were incubated in DNase freeRNase (100g/t1 in 2X SSC) at 37°C for 1 hour. This removes any RNAtranscripts which are homologous to probe sequence and in this way aids inreducing background signal. Slides were then rinsed for two minutes in each ofMaterials and Methods 41four changes of 2X SSC at room temperature, dehydrated in 75%, 85%, and95% ethanol and allowed to air dry. Chromosome DNA was denatured byimmersing slides individually in 70% formamide 2X SSC at 70°C for exactly 2minutes and immediately transferring to cold (-20°C) 70% ethanol. Slides wereagain dehydrated through an ethanol series and allowed to air dry.Hybridization mixture(3Ob’l) was applied to each slide, covered with a glass22mm x 50mm coverslip, sealed with rubber cement and incubated at 37°Covernight in a humidity chamber.Materials and Methods 42/\\/ -/Denature/ ‘__\\/ -,/ Chromosome/ preparationDNA lnseRecombinantcosmid DNANick translate/Cell cultureHarvestRNase treatment/Biotin—labelledDNADenature7Hybridize/ /1mm un o cyto chemicaldetection/ /MicroscopyFigure 5 An overview of fluorescent in situ hybridization procedures.Materials and Methods 432.7.2 Post-hybridization conditionsSlides hybridized with cosmid and alpha satellite probes were washed in 65%formamide 2X SSC at 43°C for 20 minutes. Slides hybridized with Alu PCRproduct were washed in 65% formamide 2X SSC at 37°C for 20 minutes. Eachslide was then washed twice in 2X SSC at 37°C for 4 minutes and transferredto 0.1M phosphate buffer (pH 8.0), 0.05% Tween-20.2.8 Immunocytochemical detectionHybridization signals were detected with streptavidin-fluoresceneisothiocyanate (SA-FITC) (Figure 6A). Slides were initially incubated in 6Oilof 5% BSA (in 0.1M phosphate buffer (pH 8.0), 0.05% Tween-20) at roomtemperature for 5 minutes to block non-specific SA-FITC binding. Plasticcoverslips were removed, excess BSA was shaken off and 601il of SA-FITC,4.5ng/l in 5% BSA was added to each slide. Slides were covered with plasticcoverslips and incubated at 37°C for 45 minutes in a humidity chamber. Afterincubation, slides were rinsed in 0.1M phosphate buffer (Ph 8.0), 0.05%Tween-20 three times at room temperature for two minutes each rinse. Toamplify the signal a second layer of FITC was added (Figure 6B). To blockany extraneous antibody binding, slides were incubated in 601.Ll 5% goat serum(in 0.1M phosphate buffer (pH 8.0) 0.05 % Tween-20) at room temperatureMaterials and Methods 44for 5 minutes. Plastic coverslips were removed, excess goat serum was shakenoff, and 6O1 of biotinylated anti-streptavidin antibody (Vector Laboratories)7.4ng/d in 5% goat serum was added to each slide. Slides were covered withplastic coverslips and incubated at 37°C for 45 minutes in a humidity chamber.Slides were rinsed three times in 0.1M phosphate buffer (pH 8.0), 0.05%Tween-20 at room temperature. A second incubation in BSA and SA-FITCfollowed by phosphate buffer rinses was performed and slides were mountedin antifade medium with propidium iodide (Johnson and de C. NogueiraAraujo, 1981). Coverslips were sealed with nailpolish and slides were stored inthe dark at 4°C.Materials and Methods 45A._____Botbi4ot.d prob. DNAUnamplified signal•x x••%41/• 1/ \\\\ s.’•B._ __ __ __BlctinyLot.d proD. DNAGhromeaomol DNAAmplified signalKEY• FITCBiotinyated anti—streptavidinantibody(Fab fragment)F)TC—StreptavidinBiotinFigure 6 Immunocvtochemical detection of hybridization signal. A)Unamplified signal; the biotinylated probe DNA is detected with streptavidinFITC. B) Amplified signal; streptavidin-FITC signal is amplified by applyinganti-streptavidin antibody and re-conjugating with a second layer ofstreptavidin-FITC.Materials and Methods 462.9 Microscopy2.9.1 Epifluorescence microscopeSlides were initially examined with a Carl Zeiss epifluorescence IIIRSphotomicroscope equipped with a 200 watt mercury arc lamp. Excitation anddual wavelength detection were performed using a KP 490 excitation filter, a510 beam splitter, and an LB 530 barrier filter. A Carl Zeiss 100X (numericalaperture 1.3) Planachromatic oil immersion objective lens was used. Interphasenuclei were scanned initially to determine if the hybridization had succeeded.Further image scanning and analysis was perfonned using a Bio-rad MRC 500confocal scanning laser microscope.2.9.2 Confocal microscopeImages were recorded with the Bio-rad MRC 500 confocal scanning lasermicroscope (CSLM) system equipped with an argon ion laser operating underBiorad MRC-500/600 CSLM software version 4.56. The CSLM from Bio-radwas fitted to a Carl Zeiss axiophot microscope with epifluorescence mode.Excitation and dual wavelength detection were performed with the A1/A2filter block combinations. The Al filter combination consists of a 514 DF 10exciter filter for excitation with the 514 nm line of the Ar+ ion laser(operating with neutralizing filter I; 10% of total power or 1 milliwatt) and aMaterials and Methods 47DR 527 LP dichroic reflector. The A2 filter combination consists of a DR 565LP dichroic reflector for the separation of the red propidium iodide, and thegreen FITC fluorescent light, and two barrier filters, an EF 600 LP filter whichis the “red channel” and a 540 DF 30 filter which is the “green channel”. ACarl Zeiss 100X (numerical aperture 1.3) oil immersion objective lens wasused. Digital image processing was performed using MRC 500/600 confocalmicroscope operating software CM program version 1.22 and consisted ofnoise reduction filtering through averaging or KALMAN filtering during imageacquisition, and eventual contrast enhancement by subtraction of the mean ofthe background and scaling of the remaining image. Merging of the twoimages obtained by dual wavelength detection of the FITC and propidiumiodide signals was performed through the MERGE command.2.10 Image storage and photographyAll original and processed images were archived on 3.5 inch computerdiskettes. Individual FITC and propidium iodide images as well as mergedimages were stored independently so that further image processing andphotography was possible. Colour photographs were taken from a Mitsubishi(resolution 768 X 512) colour monitor with 160 ASA colour film (AGFA).48Chapter IIIRESULTS3.1 Cosmid DNA re-annealing conditionsIn order to utilize entire cosmids as hybridization probes, for in situhybridization to metaphase chromosomes, it is necessary to block anyrepetitive sequences present within the cosmids prior to application todenatured chromosomal DNA (Landegent et al. 1987). Suppression of therepetitive content allows for both site specific hybridization and site specificdetection of the signal. To suppress these repetitive sequences cosmid DNA isallowed to partially anneal in the presence of competitor DNA. Unlabelledsheared human placental DNA was combined with the biotinylated cosmidDNA and denatured, the probe mixture was allowed to partially anneal at37°C to enable highly and moderately repetitive sequences to re-anneal(Lichter et al. 1988a; Lengauer et al. 1990). The probe mixture was then readyto be applied to the denatured chromosomal DNA.To establish appropriate re-annealing times, an estimate of repetitive sequencecontent was determined. This was achieved by observing the number andResults 49Figure 7 Agarose gel electrophoresis of an EcoRI restriction digest of cosmids11E1 and 24E10. Cosmids 11E1 and 24E10 were completely digested with therestriction enzyme EcoRI. The DNA fragments were size separated byelectrophoresis through a 0.8% agarose gel. The ethidium bromide stained gelwas photographed under ultraviolet light. The size of ) DNA fragments fromtop to bottom are as follows: 20.0, 9.3, 7.0, 4.2, 3.7, 2.8, 2.3, 2.0, 1.5, 1.0, 0.56,0.26 kb.Results 50Figure 8 Repetitive DNA content of cosmids 11E1 and 24E10. Cosmids 11E1and 24E10 were completely digested with the restriction enzyme EcoRI,separated by electrophoresis through a 0.8% agarose gel, and Southernblotted. The membrane was probed with [alpha 32P]-dATP labelled totalhuman DNA and Kodak XAR and XRP film was exposed. This is a reverseimage with light areas representing sites of hybridization.Results 51intensity of EcoRI bands, produced by complete restriction enzyme digestionof cosmid DNA, which hybridize total human DNA.The photograph of the EcoRI restriction digest of cosmids 11E1 and 24E10(Figure 7) can be compared to the autoradiograph showing the repetitiveDNA content of the cosmids (Figure 8). From this comparison a relativecomposition of repetitive DNA content can be estimated. Cosmid 11E1contains four EcoRI bands which hybridize with total human DNA with low tomoderate intensity. Ten minutes of re-annealing time was required to suppressthis repetitive content and acquire a specific hybridization signal. Cosmid24E10 contains four EcoRI bands which hybridize with total human DNA withhigh intensity. Fifty minutes of re-annealing time was necessary to block thislevel of repetitive content and acquire a specific hybridization signal.3.2 Mu element mediated PCRDNA was amplified using the A1S primer (Brooks-Wilson et a!. 1990) which iscomplementary to the extreme 3’ end of 40% of Alu consensus sequences.The template DNA used included total human DNA, somatic cell hybrid706B6-C117 DNA which contains an intact chromosome 8 as the only humancomponent, and somatic cell hybrid 706B6-C117-S12 DNA which contains 8pResults 52as the only human component. Amplification was achieved using 1.25U ofCetus Taq polymerase and a Perkin Elmer Cetus Thermocycler. The Alu PCRproducts produced were comprised of a variety of sizes ranging from 50 basepairs to 25 kilobase pairs. These products were generated by amplificationbetween two oppositely oriented and appropriately spaced Alu elements. Inthis way inter Alu DNA was amplified without amplifying Alu sequence, withthe exception of the primer sequence itself.The Mu PCR products generated using somatic cell hybrids 706B6-Cl17 and706B6-C117-S12 DNA as the template should be comprised solely of humanchromosome 8 and 8p inter Mu DNA respectively (Lengauer et al. 1990). It isalso recognized that some level of repetitive DNA will make up a portion ofthe product mixture since repetitive elements lying between oppositelyoriented Mu elements will be amplified. Size separation of synthesis productsthrough a 0.8% agarose gel stained with ethidium bromide produced a patternwhich was characteristic of each somatic cell hybrid and its particular DNAcontent with common bands likely representing the common human DNAcontent of the somatic cell hybrids. These characteristic patterns or Mu PCR“fingerprints” can be readily distinguished.Results 53Figure 9 Mu element mediated PCR “fingerprints”. Total human inter MuDNA and somatic cell hybrid inter Mu DNA was generated by Alu-PCRamplification and separated by electrophoresis through a 0.8% agarose gel.The ethidium bromide stained gel was photographed under ultraviolet light.Results 543.3 In situ hybridization banding (ISHB) of patient’ s chromosomesMu PCR products which are produced by the amplification of inter Musequences using total human DNA as template, can be hybridized tometaphase spreads of human chromosomes without previous blocking ofrepetitive sequences. In this way a banding pattern is produced which can beused to identify individual chromosomes. Since Mu elements are foundprimarily in Giemsa negatively staining regions of the chromosomes,(Manuelidis and Ward 1984; Korenberg and Rykowski 1988) the bandingpattern produced is similar to conventional reverse-banding (Baldirii et al.1991). Mu sequences are underrepresented in the centromeric region orconstitutive heterochromatin of chromosomes (Manuelidis and Ward 1984)with a lack of amplification of DNA from these regions resulting in somemodification of the banding pattern produced. There is an absence offluorescence at the centromeres of all chromosomes and in addition there isalso a lack of fluorescence in the heterochromatic regions of chromosomes 1,3, 9, and 16.Results 55Figure 10 In situ hybridization banding of patient’ s chromosomes. Partialmetaphase of the inversion duplication (8p) patient ‘ s chromosomes showingreverse-banding pattern generated by hybridization of Alu PCR amplified totalhuman DNA. Alu PCR product was hybridized to metaphase spreads ofpatient chromosomes without prior pre-amiealing in the presence ofcompetitor DNA.Results 563.4 In situ hybridizationCompetitive in situ suppression (CISS) hybridization conditions were used toproduce 8p and cosmid specific hybridization signals (Lichter et al. 1988a).The competitor DNA used was salmon sperm DNA to block highly repetitivesequences and human placental DNA to suppress highly and moderatelyrepetitive sequences. Re-annealing times for Alu-PCR generated probes weredetermined empirically with 30 minutes re-annealing being adequate toproduce a chromosome specific signal with the Alu-PCR painting of 8p.Cosmid pre-annealing times were determined as described in section 3.1.The results of hybridization of the Alu-PCR chromosome painting probe andcosmid probes, to patient metaphase spreads are shown in the followingfigures.Results 57Figure 11 Painting of 8p using Alu-PCR products from a somatic cell hybrid.This figure shows the hybridization of the Mu element mediated PCR productfrom the hamster-human hybrid cell line 706B6-C117-S12 whose sole humancomponent is 8p. The Alu-PCR product has been labelled with biotin and isdetected with FITC. The chromosomes have been counterstained withpropidium iodide. The entire p arm of both the normal and inversionduplication chromosome 8 are uniformly labelled by the AIu-PCR product.Results 58Figure 12 In situ hybridization of cosmid 11E1. This figure shows thehybridization of both the chromosome 8 centromeric probe D8Z1 and cosmid11E1 from the D8S7 locus. Both the centromeric probe and the cosmid probehave been labelled with biotin and are detected with FITC. The chromosomeshave been counterstained with propidium iodide. Two chromosomes arelabelled by D8Z1 at the centromere but only one of these is labelled bycosmid 11E1 at the telomere. The inversion duplication chromosome 8, clearlyidentified by the centromeric probe and by its longer short arm, is missing thetelomeric signal.Results 59Figure 13 In situ hybridization of cosmid 24E10. This figure shows thehybridization of both a chromosome 8 centromeric probe D8Z1 and cosmid24E10 from the D8S 133 locus. Both the centromeric probe and the cosmidhave been labelled with biotin and are detected with FITC. The chromosomeshave been counterstained with propidium iodide. Two chromosomes 8 arelabelled at the centromere. The inversion duplication 8p chromosome, clearlyidentified by the centromeric probe and by its longer short arm, shows twosites of hybridization of the cosmid probe where as the normal chromosome 8has only one site of hybridization.Results 603.5 Cosmid 24E10 localizationThe duplication of 8p has been defined cytogenetically to involve region8p12-*23.l. Cosmid 24E10 from the D8S133 locus has been mapped using asomatic cell hybrid mapping panel (Wagner et al. 1991) to interval 8cen-*21.3.Using the hybridization results along with the mapping interval established byWagner et al. cosmid 24E10 can be more specifically localized to interval8pl2-2l (Figure 14).23.3 r23.223.12221.321.221.11321.121.221.3Results 616p12 — 23.1region involved in duplication24E10 localization1211.2 J cosmid 24C10 localization11.22by hybrfd mapping panel22.122.222.32324.124.224.3I8Figure 14 Localization of cosmid 24E10.Results 623.6 MicroscopyInitial scanning of all slides was performed with a Carl Zeiss IIIRSepifluorescence photomicroscope equipped with a 200 watt mercury arc lamp.Interphase nuclei were examined initially to determine if the hybridizationprocedure had succeeded. Interphase nuclei show positive hybridization signalsvery reliably and are therefore a good starting point for analysis. After positivehybridization signals are viewed on the interphase nuclei, the hybridization wasconsidered to have succeeded and the analysis was extended to metaphasechromosomes. Since hybridization signals are more difficult to detect onmetaphase chromosomes, likely because the DNA is much more condensedand probably much less accessible to the hybridization probe, metaphaseanalysis was only attempted on those slides which had a high proportion ofsuccessful signals detected on the interphase nuclei.In general, the alpha satellite hybridization signal was easily detectable onboth interphase and metaphase spreads, cosmid hybridization signal was seenon most interphase cells but seldom could be seen on metaphasechromosomes using the epifluorescence microscope, and the Alu PCRhybridization signal could be seen well on metaphase chromosomes andsomewhat less easily on interphase chromosomes likely because the signal isResults 63very diffuse in the extended chromosomes and was therefore much duller.Once the hybridization success was established images were captured andprocessed using the MRC 500 CLSM microscope and software. With theconfocal microscope cosmid signal could be detected on all interphase andmetaphase chromosomes, with the exception of those regions of the slidedamaged throughout the hybridization procedure. Cosmid 11E1 consistentlyproduced a single hybridization signal in each cell examined and cosmid 24E10consistently produced three hybridization signals in each cell examined.64Chapter IVDISCUSSION AND CONCLUSIONSThe studies presented in this thesis have focused on the use of fluorescent insitu hybridization to reinvestigate a patient with a de novo inversionduplication involving 8p (Dill et al. 1987). The characterization of thisaberrant chromosome includes determination of the origin of the extrachromosomal material, further confirmation of the inversion nature of theduplicated segment, and definitive identification of a submicroscopic deletion.An 8p chromosome painting probe mixture was generated by Alu-PCRamplification of human inter Alu sequences specific for 8p. This wasaccomplished by amplifying DNA derived from a somatic cell hybridcontaining 8pter-8cen as the sole human component. Hybridization of thisprobe mixture confirmed the cytogenetic characterization of the origin ofduplicated material. Both the normal and aberrant chromosomes p arms wereuniformly highlighted by this probe (Figure 15) providing direct evidence thatthe duplicated material originated from 8p, rather than elsewhere in thegenome.Discussion and Conclusions 65Cosmid 24E10 from the D8S133 locus was selected as a hybridization probebased on its localization to region 8cen-’8p21.3 (Wagner et al. 1991), whichcontains a region believed to be involved in the duplication. Hybridization ofthis cosmid plays a two-fold role in the characterization of this aberrantchromosome. Its primary role is to demonstrate, by direct hybridization, theinvolvement of the D8S 133 locus in the duplication of the short arm and itssecondary role is to provide further evidence for the “mirror” nature of thisduplication. Upon hybridization the cosmid labels two sites on the aberrantchromosome (Figure 15) providing direct evidence for a duplication of theshort arm. A hybridization pattern consistent with an inversion or “mirror”duplication is seen on the aberrant chromosome. In addition, the cytogeneticcharacterization of this duplication along with the hybridization results haveallowed cosmid 24E10 to be more specifically localized to interval8pl2-+.3.Previous Southern blot dosage analysis of the patient ‘ s DNA has shown thatthe patient is monosomic at the D8S7 locus (Dill et al. 1987). This wasinterpreted to be a result of the generation of the rearranged chromosome.Therefore, the rearranged chromosome was lacking the D8S7 locus and thuswas a duplication-deficiency chromosome. However, without direct evidence, itremained uncertain whether the deletion was in fact on the inversionduplication chromosome or on the cytologically normal chromosome and theDiscussion and Conclusions 66possibility that the normal chromosome 8 is polymorphic for a null allele atthe D8S7 locus could not be excluded. Hybridization of a cosmid from theD8S7 locus to patient metaphase chromosomes provides definitive evidencethat the aberrant chromosome 8 is deleted at this locus. The cytologicallynormal chromosome 8 has a single site of hybridization whereas the inversionduplication chromosome completely lacks a site of hybridization (Figure 15).This result therefore confirms the existence of a submicroscopic deletion ofthe aberrant chromosome.Discussion and Conclusions 67NORMAL 8 INVDUP 8Figure 15 Ideogram of normal chromosome 8 and inversion duplicationchromosome 8 indicating sites of hybridization produced with the Alu-PCRDainting orobe and cosmids 11E1 and 24E10.23.323.223.1Mu PCRMu PCRp.oduct]1221.1 j 1 124db021.221.Zj -2223.12221.3 r21.221.1124Eb01211.211.111.1________________11.2211.2111.23121321.121.221.322.122.2F —22.2232221.321.221.11211.211.1I 1.1__________________11.2211.2111.23121321.121.221.322.122.222.32324.124.224.2 J24.124.224.3Discussion and Conclusions 68Of the inversion duplication 8p patients reported to date (Weleber et al. 1976;Rethoré et al. 1977; Taylor et al. 1977; Mattel et al. 1980; Poloni et al. 1981a,1981b; Jensen et al. 1982; Fryns et al. 1985; Dill et al. 1987; Kleczkowska et aL1987; Nevin et al. 1990; Gorinati et al. 1991; and Mitchell et al. 1991), mostpresent as trisomy for a region ranging from8pl2-+3.3The occurrence of adeletion in this type of chromosomal abnormality is not uncommon.Monosomy for the distal region of chromosome 8 has been demonstrated bystandard cytogenetic banding techniques (Jensen et al. 1982; Mattei et al.1980; Weleber et al. 1976; Rethoré 1977; Gorinati et al. 1991). Of theinversion duplication patients without a cytologically evident deletion, adeletion has been demonstrated at the molecular level by Southern blotdosage analysis using a probe for defensin 1 located at 8pZ3 (Mitchell et al.1991) and a probe for the anonymous locus D8S7, localized to 8p23-’8pter(Dill et al. 1987).Heterozygous deletions are difficult to identify by Southern blot analysisbecause heterozygosity is manifested only as the change in intensity of a bandand often is difficult to discern. With fluorescent in situ hybridization,heterozygosity is readily apparent as the total absence of signal on onehomologue with the presence of a signal on the other homologue serving as anDiscussion and Conclusions 69internal positive control. Since the hybridization efficiency of larger probes ishigh, deletions can be comfortably identified by analysis of just a few cells. Asa distal deletion may be a common occurrence in this type of chromosomalanomaly, investigation of those cases which have not reported such adeficiency, using fluorescent in situ hybridization and locus specific probes,may result in the discovery of a submicroscopic deletion.The clinical phenotype of patients with an inverted duplication of 8p showsome common features but also demonstrates interpatient variation. Thephenotypic effects of trisomy of various segments of chromosome 8 have beenanalyzed by Rethoré et al. (1977) and Kleczkowska et al. (1987). Theseanalyses have helped to categorize clinical signs which are common to trisomyof 8p - the most common of which include dysmorphic features, vertebralanomalies, and severe mental deficiencies, all of which are seen in the patientwho is the focus of this study. The breakpoints reported for this chromosomalanomaly vary, ranging over the region 8pi2-23.3. The variation in clinicalsymptoms may result, in part, from the differences in these breakpoints.The origin of inversion duplication chromosomes is uncertain. Severalmechanisms have been proposed for the production of such a chromosomeDiscussion and Conclusions 70either involving unequal interchange between homologous chromosomes,between chromatids of one chromosome, or between strands of one DNAduplex. Taylor et al. (1977) suggest a mechanism where two breaks occur in adonor strand and one break occurs in a recipient strand, with the donatedDNA inserted in the recipient strand in an inverted orientation (Figure 16).This mechanism retains a normal telomeric region, however it does notaccount for the loss of distal material. Two possible mechanisms have beenproposed by Mattei et al. (1980). In the first, a break in the short arm ofchromosome 8 would be the primary event leading to the production of adicentric chromosome after replication. In the second, an unequal exchangebetween short arm chromatids followed by an inversion would produce adicentric chromosome. This would be followed, in both cases, by anaphasebreakage of the dicentric chromosome and telomeric restitution resulting in ainversion duplication chromosome with a distal deficiency. This mechanism iscompatible with the symmetry seen in these inversion duplicationchromosomes as well as accounting for the loss of material distal to the breakor unequal exchange.Discussion and Conclusions 71r23.22221.321.221.11211.211.22il_III I___________121321.124.2243 JDONOR CHROMOSOME21.121.221.324.124.224.3 JRECP!ENT CHROMOSOMEFigure 16 A proposed mechanism for the generation of an inversionduplication chromosome. Two breaks occur in the donor strand and one breakoccurs in the recipient strand, with the donated DNA inserting in an invertedorientation.I23.3 r23.2 .. ..23.1 J 23.1rcc\\%\\23 1_,//) 211.211.111.1_______________11.2111.2211.23 I1221.221.322.122.222.322.122.222.32324.1Discussion and Conclusions 72A mechanism involving an aberrant recombination event to yield a dicentricchromosome joined at the centre of symmetry of the inversion duplication isfavoured by Dill et al. (1987) and Weleber et al. (1976). The aberrantrecombination would be followed by chromosome breakage and telomererestitution (Figure 17). A chromosome of this type would have a region ofduplication, whose limits are determined by the point of chromosomebreakage, as well as a distal deficiency determined by the placement of theaberrant recombination. This mechanism requires only a single primaryrecombination anomaly of an aberrant U-type exchange.Alternative mechanisms have been suggested (Gorinati et al. 1991; Mitchell etal. 1991) involving a U-type exchange in a germ cell heterozygous, de novo orby descent, for a paracentric inversion (Figure 18). A chromosome of this typewould have a region of duplication, as well as a deletion with the extent ofboth determined by the placement of the U-type exchange within theinversion. This chromosome would also have a normal distal telomeric region.While this mechanism would maintain the chromosome 8 telomere it requirestwo abnormal events, paracentric inversion formation and U-type exchange.Discussion and Conclusions 73Such inversions have not been reported in parents of inversion duplicationpatients indicating that both events occur de novo.Discussion and Conclusions 74END TO ENO”jRAPNASEFigure 17 A proposed mechanism for the generation of an inversionduplication chromosome. An aberrant recombination event yields a dicentricchromosome. Subsequent anaphase breakage produces an inversionduplication chromosome.Discussion and Conclusions 75JIli]UJ1)I ii1;Figure 18 A proposed mechanism for the generation of an inversionduplication chromosome. A paracentric inversion followed by a U-typeexchange produces an inversion duplication chromosome.I j >zi•i_ iiiriiiN N N NN N—EA/,NNNNNNNtNN — N— N Na.I________zEQJDiscussion and Conclusions 76Intact telomeres have been shown to be essential for the stability ofchromosomes, with chromosomes lacking telomeric sequence formingdicentric, ring and other unstable forms (Blackburn 1991). Recognition ofexisting telomeric sequence, by telomerase, primes the addition of telomeresequence and is thought to balance the loss of telomeric sequence due toDNA replication (Blackburn et al. 1991). Recent molecular studies (Morin1991) indicate that telomeres can be reconstituted at deletion breakpoints(Wilkie et al. 1990) with only minimal complementarity of 2 to 4 nucleotidesto the telomerase RNA template. Therefore, maintaining an intact telomere isnot an essential feature of a proposed mechanism.At a molecular level, any two members of the numerous families of repeatsequence could undergo unequal and maloriented recombination if one wasoriented towards the centromere and the other towards the telomere. Giacloneet al. (1992) have proposed possible common sequence motifs atrearrangement sites and suggest a possible mechanism which juxtaposes thesesites and mediates sequence specific breakage and recombination.4.1 Future studiesFurther study of patient G.S. ‘s chromosomes could include the use of multiplelabelling and detection fluorescent in situ hybridization systems for theDiscussion and Conclusions 77simultaneous visualization of two or more probes, this would provide moredirect evidence of the inverted nature of the duplicated segment. Upondetection, probes representing either end of the duplicated segment, anddetected with different fluorochromes, would produce an inverted signalpattern. For example, probes detected with FITC and Texas red could producea signal pattern of green, red, green, red on a tandemly duplicated segmentand a signal pattern of green, red, red, green on an inverted duplicatedsegment. In addition, an analysis of the telomeric region of the novelchromosome, using fluorescent in situ hybridization with telomere-associatedsequences, could determine if the telomere differs from a normal chromosome8 telomere.Inverted tandem duplications involving the short arm of chromosome 8 may bea non-randomly occurring de novo structural aberration in man. The futurestudy of these inversion duplication chromosomes should include an analysis ofthe rearrangement breakpoints in the search for a causative mechanisms. Thiscould include the isolation and sequencing of regions flanking therearrangements breakpoints as was done by Giacalone et al. (1992) whoisolated and sequenced the rearrangement breakpoints involved in aconstitutional X/autosome translocation and Kremer et al. (1991) who utilizedfluorescent in situ hybridization with YAC clones to isolate the DNA sequenceDiscussion and Conclusions 78which spans the fragile X region. 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USA 78:7059-7063."@en ; edm:hasType "Thesis/Dissertation"@en ; vivo:dateIssued "1992-05"@en ; edm:isShownAt "10.14288/1.0086865"@en ; dcterms:language "eng"@en ; ns0:degreeDiscipline "Genetics"@en ; edm:provider "Vancouver : University of British Columbia Library"@en ; dcterms:publisher "University of British Columbia"@en ; dcterms:rights "For non-commercial purposes only, such as research, private study and education. Additional conditions apply, see Terms of Use https://open.library.ubc.ca/terms_of_use."@en ; ns0:scholarLevel "Graduate"@en ; dcterms:title "Characterization of an inversion duplication of human chromosome 8 by fluorescent in situ hybridization"@en ; dcterms:type "Text"@en ; ns0:identifierURI "http://hdl.handle.net/2429/3323"@en .