Chromosome Analysis Chromosome Analysis

Chromosome Analysis The best whole genome analysis techniquecurrently available is .GTG-banding of Metaphase chromosomesDiagnostic Limits of ConventionalCytogenetic AnalysisChromosome Analysis G-banding provides a visual examination of theentire genome It therefore provides the best coverage but not thebest resolution Banding resolution differs from preparation topreparationHow Does Banding Resolution Impact Diagnostic Ability ? “Obvious” Aneuploidies & Rearrangements shouldbe easily diagnosed The smaller the region of gain/loss, the harder it isto detect At Absolute best, imbalances in the realm of 2-5Mbmay be detected Most banding resolutions will allow detection ofgains/deletions of 5Mb Absolute best is dependent on banding resolutionHow Does Banding Resolution ImpactDiagnostic Ability ?Molecular CytogeneticsExpanding the resolution of conventional cytogeneticanalysis Small & Subtle aberrations may be missed Depends on Banding Resolution & Specimen Preparation Most clinical labs strive for 550 band level resolution inpostnatal studies and greater than 400 bands in prenatalstudies. The resolution in bone marrows and products ofconception are often at or below the 400 band level Make do with what you have and accept the limitations Application of the techniques of Molecular Biologyto cytogenetic preparations1

FLUORESCENCE IN SITU HYBRIDIZATIONClinical Applications of MolecularCytogenetics Molecular cytogenetic techniques provide a waytodetectcomplicated,crypticandsubmicroscopic rearrangements that remainundetected or undecipherable by conventionalcytogenetic analysisFISH FISH is a physical DNA mapping technique in whicha DNA probe labeled with a marker molecule ishybridized to chromosomes on a slide, andvisualized using a fluorescence microscope The marker molecule is either fluorescent itself, oris detetcted with a fluorescently labeled antibody.HYBRIDIZATION STEPSChromatin CompactionProbe with fluorochrome or haptenGeneration of Single-StrandedDNA by DenaturationApplication ofDNA ProbeProbe recognizesTarget DNASequencesProbe hybridizesTo Target DNASequencesMetaphase chromosome is compacted into a structure that is 50,000 times shorter than itsextended lengthClassification of Chromosomal SequencesBeta satelliteAlpha satelliteClassical satelliteTelomeric sequencesUnique gene sequencesPartial chromosome paintsFISH APPLICATIONS Gene MappingChromosome IdentificationAneuploidy DetectionSexing for X-Linked diseasesMarker chromosome IdentificationTotal chromosome AnalysisTranslocation AnalysisUnique Sequence DNA DetectionMicrodeletion Syndrome AnalysisGene Amplification AnalysisMouse Chromosome ResearchWhole chromosome paints2

GENE MAPPINGFISH TECHNIQUES Metaphase FISHInterphase FISHReverse FISHMulti-color FISH (M-FISH; SKY)PRINSFiber FISHCGHDNA Arrays (Chip technology)Partial Karyotype of 133.333.3343435.135.135.355add(5)(q35)add (5)(q35)Microdeletion SyndromesChromosome 5Chromosome 11wcp 11wcp 5 Deletions of a megabase or so of DNA that aremost often too small to be seen under themicroscope Produce well defined contiguous genesyndromes which demonstrate superimposedfeatures of several different mendeliandiseases(X-linked or autosomal) Defined by high resolution banding or molecularcytogenetic techniques3

Microdeletion Studies Using FISHSyndromeChromosome LocationProbe/Gene 5S10Williams7q11.23ElastinCri du lli SyndromeAngelman Syndrome55.jpgFrom Medical Genetics (Jorde, Carey, Bamshad, White; 2nd Ed.)NormalChr 15DeletedChr 15Prader Willi Probe with controlchromosome 15 probe4

NormalChr 7DeletedChr 7Williams Probe with ControlChromosome 7 ProbeDeletedChr 22NormalChr 22Di George Probe withControl Chr 22 ProbeFISH on Interphase Nuclei isUseful in Specific Clinical Situations Prenatal diagnosis aneuploidy screening byFISH looks at interphase nuclei derivedfrom chorionic villi or amniocytes Preimplantation Genetic Diagnosisaneuploidy screening by FISH looks atinterphase blastomere nuclei5

Interphase FISH vs MetaphaseFISH Prenatal diagnosis aneuploidy screening by FISH looks atinterphase nuclei derived from chorionic villi or amniocytes Preimplantation Genetic Diagnosis aneuploidy screening byFISH looks at interphase blastomere nucleiFromMetaphaseToInterphaseChromosomes Enumeration by RapidPrenatal Interphase FISH Trisomies 13, 18 & 21 and Monosomy X are themost common aneuploidies related to maternal ageor fetal abnormality Routine chromosome analysis take 7-10 days Prenatal Interphase FISH provides a rapid way toscreen for the common aneuploidies in unculturedamniotic fluid cells in about 1-2 daysInterphase AmniocyteXDisomy 21 FemaleYChromosomes Enumerationin Chorionic Villi andAmniocytes by RapidPrenatal Interphase FISHBenefits of Prenatal Interphase FISH21Trisomy 21 Male Trisomies 13, 18 & 21 and Monosomy X are the most commonaneuploidies related to maternal age or fetal abnormality Routine chromosome analysis take 7-10 days Prenatal Interphase FISH provides a rapid way to screen for thecommon aneuploidies in uncultured amniotic fluid cells in about 1-2days Reduces emotional burden on the patient and/or physician in the face of anincreased risk for chromosome abnormalities following an abnormal screeningresult¾Opportunity to reduce anxiety through earlier decision making6

Sensitivity of PrenatalInterphase FISHAbnormalityPreimplantation y 1398.6%100%100%99.98%99.8% PGD is a very early form of prenatal diagnosisAneuploidy 1899.5%100%100%Trisomy 21100%100%100%100%45,X100%99.98%98.5%100%Other SexChrAll detectable100%100%100%100% 99.6%99.98%99.8%99.96% Oocytes or embryos obtained in vitro throughassisted reproductive techniques are biopsiedPolar bodies for the oocytesBlastomeres for the embryosFrom Tepperberg et al. Prenatal Diagnosis 2001; 21:293-301 Study looked at 5197 pregnancies Review of Lit includes 30,000 pregnancies Only Embryos shown to be free of the diseaseunder consideration are subsequently used fortransferPreimplantation GeneticDiagnosisBiopsy Method Generate Oocytes/embryos in vitro by ARTBiopsy Polar Body/1-2 cells from embryosFISH or PCR for genetic diagnosis Patients Polar Body Cleavage StageRepeated terminationsMoral or religious objections to terminationRepeated miscarriages due to chromosome abnormalityInfertile couples BlastocystPolar Body BiopsyDay 3 - Cleavage Stage Used by few groups in USA Used by majority of groups Need the first and second polar body Biopsy at 6-10 cell stage Labour intensiveUsed by majority of groups Only maternal chromosomes examined Blastomeres totipotent 1-2 cells for analysisFrom Veeck – Atlas of EmbryologyFrom Veeck – Atlas of Embryology7

Embryo BiopsyEmbryo Biopsy – Acid TyrodesCourtesy of Kathleen Miller, RMA of NJCourtesy of Kathleen Miller, RMA of NJAneuploidy Analysis of Embryos forChromosomes 13, 16, 18, 21 & 22Multiple Hybridizations AddDiagnostic PowerChr. 18Chr. 13Chr. 16Chr. 21Trisomy 22Chr. 22Chr. 13Chr. 18Chr. XChr. 21Chr. YChr. 13Chr. 16Chr. 18MosaicismMosaicismPost-Zygotic Non–DisjucntionPost-Zygotic Non–DisjucntionChromosomes1517XChr. 21Chr. 22Chromosomes13161821228

TelomeresMosaicismPost-Zygotic Non–DisjucntionChromosomes1316182122 Highest concentration of genes of any chromosomalregion – therefore sub-microscopic deletions andduplications would have a significant impact Increased genetic recombination at telomeres Male rate higher than female for most chromosomes Telomeres play a critical role in chromosome pairingat meiosisFISH using Subtelomere ProbesPericentric Inversion5p 13qPericentric InversionFISHThe Clinical Phenotype Guides the Choice of FISH TestPrader-WilliSyndrome ?Williams Syndrome ?Mother5p 5qProband9

The Clinical Phenotype Guides the Choice of FISH TestHow do we determine theorigin of this markerchromosome?Options: Sequential trial & error withcentromeric probes Sequential trial & error withwhole chromosome paintsMarkerWhat FISH test do we do in this case ?Detection of Partial Aneuploidies –An expensive FISHing Expedition REVERSE FISH FOLLOWING MARKERCHROMOSOME MICRODISSECTIONUnbalanced rearrangementsMarker chromosomesCryptic translocationsCryptic deletionsSuspected Microdeletions with nonspecific clinical abnormalities18p18pThe Need for New TechnologiesSpectral KarYotyping(18q- ; X )COMPARATIVE GENOMIC HYBRIDIZATIONCGH Identifies chromosomal gains and losses in asingle hybridization procedure Effectively reveals any DNA sequence copynumber changes (i.e., gains, amplifications, lossesand deletions) in a particular specimen and mapsthese changes on normal chromosomes(Schrök et al – Hum Genet 1997)10

CGH36.336.236.13534.334.234.133 LOSS32In situ hybridization of differentially labelled specimenDNA & normal reference DNA to normal humanmetaphase chromosome spreads.31222113HeterochromaticRegion12111112 GAIN21Specimen & reference DNA can be distinguished bytheir different fluorescent colors.222324253132414243440.50.75 1.0 1.25 1.5Chromosome 1DAPIFITCX ChromosomeDAPIFITCInvertedDAPITexasRedChromosome 9X ChromosomeY ChromosomeChromosome 9FITC Texas RedTexasRedY ChromosomeOVERVIEW OF CGH (cont)OVERVIEW OF CGHThe major steps in CGH involve:FITC Texas Red In situ hybridization of the labelled specimen &reference DNAs to normal metaphase spreads Preparation of normal metaphase spreads Washing off unbound DNA Isolation of high molecular weight DNA fromspecimen (test) and reference (normal) samples. Counterstaining metaphase spreads with DAPI Labelling of specimen & reference DNA withdifferent color fluorochromes Fluorescent microscopy to visualize & capturecolor ratio differences along the chromosomes11

Microarray MethodologyCGH MicroarrayMethodologyDNA MicroarraysControl genomicDNAArrayed clonesTest genomicDNAAutomatedanalysisCloned humanDNA (BAC/PAC)CGH Microarray MethodologyDNATrisomy 21 (47,XY, 21)CHIPTECHNOLOGY12

CGH Microarray MethodologyCGH Microarray MethodologyTrisomy 18 (47,XY, 18)Patient Clinical InfoDelayed major motor milestones, includingrolling over at 4 months, sitting withoutsupport at 8 months, and walking at 22months. He had been receiving regularphysical therapy for delay in gross motorskills. He spoke in short sentences andunderstood complex commands. Therewere no facial dysmorphisms besidesslightly cupped ears and no medicalproblems except for severe eczema.Data AnalysisROMAN ABCNAG 0120000000Normal development status at 3 yrs, exceptfor mild delay in gross motor skills andcoordination.MICROARRAYSIN CLINICAL CYTOGENETICS Precise identification of extra or missing material– Important for diagnostic and prognostic value– Important for identifying those genes causative of theclinical phenotype Single step global genome scan preventsFISHing expeditionDNA based analysis– Quality of metaphase spreads is not a consideration– Non-viable tissues are amenable to analysisBenefits of CGH/Microaray Analysis inClinical GeneticsThe ability to define more precisely thechromosomal material comprising markerchromosomes and unbalanced translocationsmay help to further define critical chromosomalregions which are associated with normal andadverse phenotypic outcomes and thusprovide prognostic information for geneticcounseling.13

Benefits of CGH Analysis in Clinical GeneticsBenefits of CGH Analysis in Clinical GeneticsIt is therefore prudent for investigators who areutilizing the newer molecular cytogenetictechniques to report their findings inconjunction with the clinical presentation sothat a comprehensive database can beconstructed.Information derived from such a databasewould directly benefit prenatally ascertainedcases of chromosomal imbalance, providingcouples with a means to make rational andinformed decisions concerning the pregnancy.In pediatric cases, such information mayprovide the parents with a realistic prognosisand be important for the clinical managementof the infant.14

Cri du chat 5p15.2 D5S23 Wolf-Hirschhorn 4p16.3 D4S96 55.jpg From Medical Genetics (Jorde, Carey, Bamshad, White; 2nd Ed.) Prader-Willi Syndrome Angelman Syndrome Normal Deleted Chr 15 Chr 15 Prader Willi Probe with control chromosome 15 probe