Introduction To Cytogenetics Part 2

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Introduction to CytogeneticsPart 2Erica Andersen, PhDSection Chief, Cytogenetics and Genomic Microarray,ARUP LaboratoriesAssociate Professor, Department of PathologyUniversity of Utah

Introduction to Cytogenetics II Structural Chromosome Abnormalities––––––Underlying MechanismsNomenclatureDeletions and DuplicationsTranslocations and Segregation MechanismsX-chromosome AbnormalitiesInversions and Recombinant Chromosomes Cytogenetics in Cancer– Hematologic malignancies overview– Cytogenetic abnormalities and nomenclature– Genetic basis of cancer: oncogenes, tumorsuppressors

Structural Abnormalities Definition: Breakage and rejoining ofchromosomes or chromosome segments May be either balanced or unbalanced Breakpoints can disrupt gene expression(within a gene or regulatory element) Can create gene fusions or affect geneexpression ( ) by position effect– Common in cancer

Mechanisms Underlying StructuralRearrangements- Errors in Recombination: exchanges between homologous,non-allelic sequences via non-allelic homologousrecombination (NAHR) Repair: double-stranded breaks that are repairedincorrectly by non-homologous end-joining(NHEJ) Replication: discontinuous replication of thelagging strand leads to invasion into otherreplication forks: fork stalling and templateswitching (FoSTes)

Structural abnormalities(Abnormal is on the right)DeletionsTerminalInterstitialReciprocal ncedRobertsonian TranslocationsBalancedUnbalanced

Structural abnormalities(Abnormal is on the omosomesRing chromosomesParacentricinversionIsochromosomes

Normal variable chromosomal features/Heteromorphisms(NOTE: generally, these are not included in the karyotype)Normal 9’sVariation in length ( or -) 1qh Yqh 13ps 9qh 16qh 21pstkVariation in position inv(2)(p11.2q13) inv(9)(p12q13) Yqsinv(9)(p12q13)9qh-

Designation of Regions, Bands, Sub-bandsIdiogramSub-bandRegion Bandp armTelomere(pter)1Centromereq arm123Telomere(qter)65432112313 24567812345Chr. 4Example: 4p15.35.35.25.101.11.21.3400550700

Differences in level of resolution by sample type350BM400-425AFPOC550-700PB

Standard Nomenclature for Karyotype DesignationGeneral designation includes: Chromosome number (count)-based on #centromeres– Expressed relative to the ploidy level Sex chromosome constitution– Use /- for acquired sex chromosome aneuploidy only List of abnormalities present– Ordered by chromosome number (sex chromosomes, thenautosomes 1-22) and abnormality type (numericalabnormalities/aneuploidies, then structural abnormalities, listedalphabetically and by arm/band, low to high) Multiple cell lines– Mosaicism: List abnormal clone(s) first, list multiple abnormalclones from largest to smallest in size– Chimerism: List recipient (individual’s karyotype) first

Common symbols and abbreviated terms(constitutional studies) add delderdicdupdniidicinsinvmarmatmospatrrob t///[ ]additional normal or abnormal chromosome (trisomy)loss of a chromosome (monosomy)added material of unknown origin, typically resulting in a loss ofmaterial distal to breakpointdeletionderivative chromosome, due to structural rearrangement(s)dicentric chromosomeduplicationde novo (not inherited)isochromosome (composed of two identical chromosome arms)isodicentric chromosome (isochromosome w/ two centromeres)insertioninversionmarker chromosome, unknown originmaternal originmosaic (multiple cell lines/clones present)paternal originring chromosomeRobertsonian translocation, a whole arm translocation betweenacrocentric chromosomestranslocationseparates clones (for mosaic karyotypes)separates clones (for chimeric karyotypes)indicate number of cells (for mosaic or chimeric karyotypes)

Structural Abnormalities Description(Illustrated by Examples) Terminal vs interstitial– add(11)(q23)– del(4)(p16.3)– dup(17)(p11.2p13) Interchromosomal vs intrachromosomal– t(9;22)(q34;q11.2)– inv(3)(q21q26.2)– ins(2)(q13p11.2p14) Whole chromosome arm rearrangements– i(12)(p10)– der(1;7)(q10;p10)– rob(13;14)(q10;q10) Combination of abnormalities– 47,XY, 8,t(8;14)(q24;q32)– der(7)del(7)(p11.2)del(7)(q22)– mos 45,X[12]/46,X,idic(X)(p11.22)[8]

Nomenclature Practice:Structural Abnormalities

Abnormal, constitutionalFemalep13p11.2

Abnormal, 2

Abnormal, constitutionalMale with Klinefelter syndromeq22q24.1q32

Abnormal, constitutional47,XXY,ins(13;12)(q32;q22q24.1)Male with Klinefelter syndromeq22q24.1q32

Abnormal, constitutional

Abnormal, constitutional45,XX,rob(14;15)(q10;q10)

Abnormal, constitutionalFemaleq11.2q23.3

Abnormal, constitutional47,XX, der(22)t(11;22)(q23.3;q11.2)Femaleq11.2q23.3

Structural al ncedRobertsonian TranslocationsBalancedUnbalanced

Some recurrent deletions and duplications1p36 del2q37 delBDMR7q11.23 2delLangerGiedion8q24 delInv dup 15*PWS/AS15q11-13del pat/mat& dup mat*TetrasomyCri du chat5p15 delWolfHirschhorn4p16.3 delBWS/RSS11p15 duppat/matWAGR11p13 del* PallisterKillianJacobsen11q24 delRubenstein-Taybi16p13.3 lle20p12 delMiller-Dieker 17p13.3delHNPP/CMT1A17p11.2 del/dupXp22.31 STS/KAL del22q11 del(VCFS)/dup* Cat-eyePhelanMcDermid22q13 delImage modified from Gardner, Sutherland and Shaffer Chromosome Abnormalities and Genetic Counseling 4th ed (2011)

Incidence of Recurrent Deletion andDuplication SyndromesSyndromeIncidenceCause1p36 deletion1:7500Terminal deletion1q21.1 deletion (distal)1:500Interstitial deletion (SD)4p-/Wolf-Hirschhorn1:50,000Terminal deletion5p-/Cri du chat1:50,000Terminal deletion7q11.23/Williams1:7500Interstitial deletion (SD)15q11q13/Prader willi1:20,000Interstitial ge/VCFS1:5000Interstitial deletion (SD)

Low copy repeats (LCRs) mediate many recurrent genomicrearrangements via NAHRKeyNAHR-prone regionsDeletion disordersDel/dup disordersLiu et al, 2012

Segmental duplication (low-copy repeat, LCR)architecture mediates recurrent CNVs/rearrangementsEmanuel and Saitta, Nat Rev Genet 2007

NAHR: misalignment and exchange occurs betweennon-allelic homologous sequences (LCRs)DxD allelic HRBalancedrecombinantsDxA non-allelic HRUnbalancedrecombinantsDuplicatedDeletedEmanuel and Saitta, Nat Rev Genet 2007

NAHR underlies many recurrent genomic rearrangementsLiu et al., 2012

Multiple techniques are employed for the detection ofdifferent cytogenetic dabs?Structuralinfo?G-bandedchromosomes3-5 Mb(550 bands)10-15%YesYesYesYesMetaphase FISH100’s kbn/aYesNoYesYesInterphase FISH100’s kb1-5%NoNoYesYesGMA10-100’s kb10-20%NoYesYesNo Sizes: kb 1x103, Mb 1x106

Structural al ncedRobertsonian TranslocationsBalancedUnbalanced

Incidence of chromosome abnormalities detected innewbornsAbnormalityRate/1000Rate (1/n)Autosomal Trisomy1.62617Sex Chromosome Aneuploidies (All)2.70375Balanced Structural Rearrangements2.04490Translocations, s0.911,099Unbalanced Structural Rearrangements0.631,587Translocations, insertions, ns, rings0.0617,184 Markers (e.g. isochromosomes)0.412,455Data from: Milunsky and Milunsky, Genetic Disorders of the Fetus, 6th Ed. (2010). Benn, Chp. 6 1/500 is a carrier of a balanced rearrangement

Effects of Translocations Constitutional carriers are at risk for infertility,recurrent miscarriage and/or birth of a child with acongenital anomaly syndrome– Most risk figures fall into the range of 0-30% for a livebornchild with an abnormality (higher end if previous child) May disrupt gene expression (breakpoint within a geneor regulatory element by position effect)– In prenatal setting and de novo, risk 6% (Warburton ‘91) Create gene fusions and affect gene expression byposition effect– Esp. in cancer ex. t(9;22) BCR-ABL1 chimeric transcript ort(11;14) CCND1 upregulation by translocation near the IGHlocus regulatory region

Pachytene configuration (quadrivalent) in the balancedtranslocation carrier/translocation heterozygoteA, B: Normal chromosomesA’, B’: Derivative chromosomesGardner, Sutherland and Shaffer. 2012

Modes of Segregation DuringGametogenesis in theBalanced Translocation CarrierOnly 2:2 alternate segregation willresult in normal/balanced gametesAll other modes of segregation resultin unbalanced gametesChromosome Abnormalities and Genetic Counseling.4th ed. Gardner, Sutherland and Shaffer. 2012

Predicting clinical outcomes for the balancedtranslocation carrierFactors that influence segregation and outcomes Location of the breakpoints, relative to chromosome size and the centromere Relative size of chromosomes involved See also Table5-4 in Gardner,Sutherland andShaffer 2012Gardner, Sutherland and Shaffer. 2012

Tertiary trisomy in the t(11;22)(q23;q11) carrier46,t(11;22)47, der(22),t(11;22)(Emanuel syndrome)Tertiary trisomy3:1 segregationGardner, Sutherland and Shaffer. 2012

Predicting clinical outcomes for the balancedtranslocation carrierFactors that influence segregation and outcomes Location of the breakpoints, relative to chromosome size and the centromere Relative size of chromosomes involved Biological consequence of associated monosomy/trisomy Least imbalanced, least monosomic is most likely to produce a viable conceptus See also Table5-4 in Gardner,Sutherland andShaffer 2012Gardner, Sutherland and Shaffer. 2012

Pedigree of a family carrying atranslocation with a large centric segmentGardner, Sutherland and Shaffer. 2012

Structural al ncedRobertsonian TranslocationsBalancedUnbalanced

Robertsonian translocations Frequency 1/1000, 95%are nonhomologous– rob(13;14) is mostcommon (1:1300) Homology andorientation of sequencesin p-arm stalks of chrs13, 14 and 21 likelyexplain relativeprevalence of rob(13;14)and rob(14;21) amongstcarriers (via NAHR)Gardner, Sutherland and Shaffer. 2012

Robertsonian translocations:Meiotic lCarrierTrisomyMonosomyModified from Gardner, Sutherland and Shaffer. 2012

Imprinted chromosomes and human diseasedue to uniparental disomy (UPD)Image from: http://carolguze.com/text/442-10nontraditional inheritance.shtmlVelissariou, Balkan J Med Gen

Risk for uniparental disomy (UPD) Risk for expression of clinical phenotype if rob chromosome contains imprintinggenes (differentially expressed genes based on parent of origin) (chrs. 14 and 15)Images modified from from Shaffer et al., 2001, Genetics in Medicine Heterodisomy: two homologous copies or segments from the same parent

Risk for uniparental disomy (UPD) Risk for expression of clinical phenotype if rob chromosome contains imprintinggenes (differentially expressed genes based on parent of origin) (chrs. 14 and 15)Images from Shaffer et al., 2001, Genetics in Medicine Isodisomy: two identical copies or segments from the same parent Risk for expression of two recessive alleles with isodisomy

Empiric risk estimates for offspring of Robertsoniantranslocation carrier Risk to have unbalancedis greater for females 10-15% forchromosomes 21 Risk for UPD is the same The risk to homologousrob carriers is 100% Very rare instances ofpost-zygotic correctionare reportedGardner, Sutherland and Shaffer. 2012

Modes of X-inactivationMorey and Avner, 2001 Most X-inactivation occurs randomly– Random X-inactivation often protects against (masks) pathogenic (recessive) mutations in females Non-random (skewed) X-inactivation may occur by chance (primary) or through cellselection (secondary)– Can lead to expression of X-linked recessive mutations in females– Can protect against an otherwise dominant-acting mutation

Non-random X-inactivation can rescue effects ofX-chromosome abnormalities in females Most structural abnormalities and some mutationslead to non-random inactivationKey Active X White Inactive X Gray * XIST*Leppig and Disteche, Semin Reprod Med, 2001

Translocation X;A in females-balanced carriers may alsobe affected, dependent on X-inactivation There is an inherent risk to the balancedfemale carrier if X inactivation is notskewed to preferentially inactivate thenormal X Risk for functional disomy (doubleexpression of X-linked genes relativeto their normal level) of thetranslocated X segment on theder(A) Risk for functional monosomy of thetranslocated autosomal segment onthe der(X)Key Active X White Inactive X Gray Autosomalmaterial hashed * XISTLeppig and Disteche, Semin Reprod Med, 2001

Structural nantchromosomesRing chromosomesParacentricinversionIsochromosomes

Recombinant chromosome arises from aparental pericentric inversion1234567165432712345677654327Image source: http://www.ucl.ac.uk/ ucbhjow/bmsi/bmsi 7.htmlrec(8)dup(8q)inv(8)(p23.1q23.1)

Cytogenetics in Cancer Information from cytogenetic testing is used to:– Establish diagnosis– Guide therapy– Predict outcome– Monitor response to therapy or engraftment postbone marrow transplant (BMT)

Basic terminology for classifyinghematologic malignancies Leukemia: cancer of the blood and/or bone marrow Lymphoma: cancer in the lymphatic tissue (nodal orextranodal) Myeloid: cells that arise and differentiate in the bonemarrow (RBC’s, platelets, WBCs: granulocytes) Lymphoid: cells that arise in the bone marrow anddifferentiate and/or function in the lymphatic system(WBC types: B-cells, T-cells, NK cells)

Blood Cell LineagesMyeloid-typediseases AML CML MDS MPDLymphoid-typediseases ALL CLL MM LymphomasImage source: s/2009/02/hematopoiesis simple1.png

Types of Chromosome Abnormalitiesin Cancer Numerical– Aneuploid: 2n - or chromosomes Monosomy or trisomy– Polyploid: 1n, 2n, 3n, 4n, etc. where n 23 chr. icationsTranslocations: balanced or unbalancedInversions Copy-neutral loss of heterozygosity (LOH)– Mitotic recombination– Mitotic malsegregation: uniparental disomy

Comparing technologies Aberrations of copy number, structureBalancedKaryotypingFISHCMA (SNP) -Aberrations of genotypeUnbalanced / Image modified from Albertson et al., 2003, Nature Genetics

Defining clonality/acquired changes inoncology studies Karyotyping:– At least two metaphase cells with the same extrachromosome, structural abnormality– At least three metaphase cells with the same chromosome loss FISH:– Abnormality observed in a percentage of cells (usually 1-5%), 200interphase FISH cells are examined Genomic microarray:– Evidence of mosaicism in the sample as shown by the copy numberand/or SNP-containing probes– Cannot determine whether multiple mosaic abnormalities representdifferent clones/evolution (clonal diversity)

Karyotyping in Cancer

e.g. Clinical Utility of Karyotype in ALLProportion of casesCytogenetic subtype distribution by ageHarrison. ASH Education Program (2013) 118-125

The Genetic Basis of Cancer

Types of genes involved in cancerCalvert and Frucht, 2002, Ann Int Med

Types of genes in cancer Oncogenes: mutant forms of genes (protooncogenes) that positively regulate cell proliferationand survival Dominant, gain-of-function type mutationsImage source: http://www.scq.ubc.ca/images/oncogeneformation.gif

Mechanisms of oncogene activation Chromosomal rearrangements(translocations, inversions)– A gene fusion creating a chimericprotein– Upregulation of gene expressionby position effect Copy number gains– Trisomy, tetrasomy, etc.– Gene amplificationImage modified from Albertson et al., 2003, Nature Genetics

Oncogene Activation by GeneFusion

t(9;22) in chronic myelogenous leukemia (CML) First chromosomal abnormalityassociated with cancer, discovered in1960 Abnormal Chr. 22 named thePhiladelphia (Ph) chromosomePhImage t0922CML.htmlder(9)922der(22)

The t(9;22)(q34;q11) reciprocaltranslocation(Proto-oncogene)

BCR/ABL1 protein is a constitutively activetyrosine kinase The N-terminal capregulates controlled ABLkinase activity Fusion to 5’ BCR– Increases cellproliferation– Inhibits programmed celldeath– Increases invasiveness– Inhibits DNA repairGoldman and Melo, NEJM, 2003

Targeted Therapy: Inhibitors of tyrosinekinase (TKIs) Imatinib mesylate (Gleevec)was the first TKI approvedby the FDA in 2001 Mechanism: Competes withATP for binding sites Inhibits progression of CMLin the majority of patients Drug resistance can developover timeBCR-ABL1 kinase inhibited by ImatinibImage ns/c/ca/Bcrabl STI 1IEP.png

Oncogene Activation by PositionEffect

c-MYC rearrangements in Burkitt lymphoma Cell of origin is a peripheralmemory B-cell c-MYC at 8q24 is a protooncogene is a transcription factorthat induces cell proliferation Immunoglobulin genes arestrongly expressed in B-cells Translocation juxtaposes c-MYCwith IG enhancers t(8;14)(q24;q32) in 75-85% cases t(8;22)(q24;q11) in 10% cases t(2;8)(p12;q24) in 5% casesIGΚ locus on 2pIGH locus on 14qIGL locus on 22qImage t0814ID1050.html

C-Myc influences the transcription of a variety ofproteins involved in the cell cycleBlum et al., Blood. 2004

Selected Rearrangements in CancerNeoplasmTranslocationPercentage ofCasesOncogeneChronic myelogenousleukemiat(9;22)(q34;q11)100% (includesvariant fusions)BCR-ABL1Acute cute lymphocyticleukemiat(4;11)(q21;q23)5-10%; 40% 1yKMT2A-AFF1Acute Acute myeloid leukemiat(8;21)(q22;q22)5-10%RUNX1T1-RUNX1Acute myeloid leukemiainv(16)(p13.3q22) kitt lymphomaMYC

Types of genes in cancer Tumor suppressors: genes that block tumordevelopment by negatively regulating cell growthand proliferation Recessive, loss-of-function type mutationsGermlineKnudson’s TwoHit itarycancersTumorImage modified from UW Cytogenetics Lab

Mechanisms of tumor suppressorinactivation Copy number losses– Monosomy– Deletions– Note: copy number loss mayin itself be pathogenic ormay unmask a recessivemutant alleleMonosomyDeletion Loss of heterozygosity(LOH)– Somatic recombination– Uniparental disomyImage modified from Albertson et al., 2003, Nature Genetics

Nomenclature in Cancer

Common symbols and abbreviated terms add ccpdelderdicdmindupiidicinsinvmarrslsdlt?///[ ]additional normal or abnormal chromosome (trisomy)loss of a chromosome (monosomy)added material of unknown origin, typically resulting in a loss ofmaterial distal to breakpointconstitutionalcomposite (clonal, but variable across cells)deletionderivative chromosome, due to structural rearrangement(s)dicentric chromosomedouble minute chromosomeduplicationisochromosome (composed of two identical chromosome arms)isodicentric chromosome (isochromosome w/ two centromeres)insertioninversionmarker chromosome, unknown originring chromosomestemline (used with clonal evolution)sideline (used with clonal evolution)translocationdesignates uncertainty (used in place of, or in front of a finding)separates clones (for mosaic karyotypes)separates clones (for chimeric karyotypes)indicate number of cells (for mosaic or chimeric karyotypes)

Case 1: CHR BM for a patient after treatment for AML showsdisease arrangement involving 11q23 (MLL/KMT2A) associated w/ a poor prognosis in AML

Case 2: AML, CHR BM reveals complex karyotype with multiplerelated abnormal clones, shows clonal .2q13.1)[4]/46-47,sl, 8,ins(11;?)(q13;?),212dmin[cp13]/46,XX[3]Complex karyotypes are associated w/ a poor prognosis in AML

Case3: CHR BM reveals trisomy 21 in a newborn male with pancytopenia(uncertain if patient has Down syndrome)47,XY, 21[20]?c Careful with abnormalities present in every cell ?constitutional DS patients have an increased risk of transient myeloid disease and ALL Trisomy 21 is a recurrent acquired change in hematologic disease Test PB lymphocytes to see whether abnormality is constitutional/clonal

Case 4: CHR BM on a patient with multiple myeloma (MM)reveals a complex karyotype44-45,XY, 19] Loss of Chromosome 17 (TP53 gene) is associated with unfavorableprognosis in MM (and virtually all other cancers)

Principles of CytogeneticsCategorical CourseIntroduction to Cytogenetics 2Erica Andersen, PhDMedical Director, Cytogenetics and Genomic Microarray,ARUP LaboratoriesAssistant Professor, Department of PathologyUniversity of Utah

Introduction to Cytogenetics II Structural Chromosome Abnormalities –Underlying Mechanisms –Nomenclature . Genetics in Medicine Risk for expression of clinical phenotype if rob chromosome contains imprinting genes (differentially exp

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