Introduction to Cytogenetics Part 2 Erica Andersen, PhD Section Chief, Cytogenetics and Genomic Microarray, ARUP Laboratories Associate Professor, Department of Pathology University of Utah  Introduction to Cytogenetics II Stru...

Introduction to Cytogenetics Part 2 Erica Andersen, PhD Section Chief, Cytogenetics and Genomic Microarray, ARUP Laboratories Associate Professor, Department of Pathology University of Utah  Introduction to Cytogenetics II Structural Chromosome Abnormalities – Underlying Mechanisms – Nomenclature – Deletions and Duplications – Translocations and Segregation Mechanisms – X-chromosome Abnormalities – Inversions and Recombinant Chromosomes Cytogenetics in Cancer – Hematologic malignancies overview – Cytogenetic abnormalities and nomenclature – Genetic basis of cancer: oncogenes, tumor suppressors  Structural Abnormalities Definition: Breakage and rejoining of chromosomes 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 gene expression (↑↓) by position effect – Common in cancer  Mechanisms Underlying Structural Rearrangements- Errors in… Recombination: exchanges between homologous, non-allelic sequences via non-allelic homologous recombination (NAHR) Repair: double-stranded breaks that are repaired incorrectly by non-homologous end-joining (NHEJ) Replication: discontinuous replication of the lagging strand leads to invasion into other replication forks: fork stalling and template switching (FoSTes)  Structural abnormalities (Abnormal is on the right) Deletions Duplications Insertions Terminal Interstitial Reciprocal Translocations Robertsonian Translocations Balanced Unbalanced Balanced Unbalanced  Structural abnormalities (Abnormal is on the right) Inversions Ring chromosomes Pericentric Paracentric inversion inversion Recombinant Isochromosomes chromosomes  Normal variable chromosomal features/ Heteromorphisms (NOTE: generally, these are not included in the karyotype) Normal 9’s Variation in length (+ or -) 1qh+ Yqh+ 9qh- 13ps+ 16qh+ 21pstk- Variation in position inv(9)(p12q13) 9qh- inv(2)(p11.2q13) inv(9)(p12q13) Yqs  Designation of Regions, Bands, Sub-bands Idiogram Sub-band 400 550 700 Region Band Telomere Example: 4p15.3 (pter) 6 p arm 5.3 1 5 5.2 43 5.1 Centromere 21 12 0 1 3 1 3 24 2 5 q arm 6 7 8 1.1 3 1 1.2 2 1.3 3 4 Telomere 5 (qter) Chr. 4 Differences in level of resolution by sample type 350 400-425 550-700 BM AF POC PB  Standard Nomenclature for Karyotype Designation General 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, then autosomes 1-22) and abnormality type (numerical abnormalities/aneuploidies, then structural abnormalities, listed alphabetically and by arm/band, low to high) Multiple cell lines – Mosaicism: List abnormal clone(s) first, list multiple abnormal clones from largest to smallest in size – Chimerism: List recipient (individual’s karyotype) first  Common symbols and abbreviated terms (constitutional studies) + additional normal or abnormal chromosome (trisomy) - loss of a chromosome (monosomy) add added material of unknown origin, typically resulting in a loss of material distal to breakpoint del deletion der derivative chromosome, due to structural rearrangement(s) dic dicentric chromosome dup duplication dn de novo (not inherited) i isochromosome (composed of two identical chromosome arms) idic isodicentric chromosome (isochromosome w/ two centromeres) ins insertion inv inversion mar marker chromosome, unknown origin mat maternal origin mos mosaic (multiple cell lines/clones present) pat paternal origin r ring chromosome rob Robertsonian translocation, a whole arm translocation between acrocentric chromosomes t translocation / separates 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/46,X,idic(X)(p11.22) Nomenclature Practice: Structural Abnormalities Abnormal, constitutional Female p13 p11.2 Abnormal, constitutional 46,XX,del(11)(p11.2p13) Female p13 p11.2 Abnormal, constitutional Male with Klinefelter syndrome q22 q24.1 q32 Abnormal, constitutional 47,XXY,ins(13;12)(q32;q22q24.1) Male with Klinefelter syndrome q22 q24.1 q32 Abnormal, constitutional Abnormal, constitutional 45,XX,rob(14;15)(q10;q10) Abnormal, constitutional Female q11.2 q23.3 Abnormal, constitutional 47,XX,+der(22)t(11;22)(q23.3;q11.2) Female q11.2 q23.3  Structural abnormalities Deletions Duplications Insertions Terminal Interstitial Reciprocal Translocations Robertsonian Translocations Balanced Unbalanced Balanced Unbalanced  Some recurrent deletions and duplications 1p36 del Wolf- Cri du chat 5p15 del *Tetrasomy Hirschhorn 4p16.3 del 2q37 del BDMR BWS/RSS 11p15 dup pat/mat 7q11.23 del Potocki- Shaffer WAGR * Pallister- Killian 11p11.2 11p13 del (WBS)/dup Langer- del Giedion Jacobsen 8q24 del 11q24 del Inv dup 15 RB1 *PWS/AS Rubenstein Smith-Magenis/ Miller-Dieker 17p13.3 del 13q14 15q11-13 -Taybi Potocki-Lupski del del pat/mat 16p13.3 del 17p11.2 HNPP/CMT1A & dup mat del/dup 17p11.2 del/dup Alagille Xp22.31 STS/KAL del 18p- 20p12 del 22q11 del 18q- (VCFS)/dup * Cat-eye Phelan- McDermid 22q13 del Image modified from Gardner, Sutherland and Shaffer Chromosome Abnormalities and Genetic Counseling 4th ed (2011)  Incidence of Recurrent Deletion and Duplication Syndromes Syndrome Incidence Cause 1p36 deletion 1:7500 Terminal deletion 1q21.1 deletion (distal) 1:500 Interstitial deletion (SD) 4p-/Wolf-Hirschhorn 1:50,000 Terminal deletion 5p-/Cri du chat 1:50,000 Terminal deletion 7q11.23/Williams 1:7500 Interstitial deletion (SD) 15q11q13/Prader willi 1:20,000 Interstitial deletion (pat)/mUPD/Me defect/mutation 22q11.2/DiGeorge/VCFS 1:5000 Interstitial deletion (SD) Low copy repeats (LCRs) mediate many recurrent genomic rearrangements via NAHR Key NAHR-prone regions Deletion disorders Del/dup disorders Liu et al, 2012  Segmental duplication (low-copy repeat, LCR) architecture mediates recurrent CNVs/rearrangements Emanuel and Saitta, Nat Rev Genet 2007  NAHR: misalignment and exchange occurs between non-allelic homologous sequences (LCRs) DxD=allelic HR Balanced recombinants DxA=non-allelic HR Unbalanced Duplicated recombinants Deleted Emanuel and Saitta, Nat Rev Genet 2007 NAHR underlies many recurrent genomic rearrangements Liu et al., 2012 Multiple techniques are employed for the detection of different cytogenetic abnormalities Balanced Sensitivity Culturing Unbalanced abs? Technique Resolution Global? (mosaicism) required? abs? Structural info? G-banded 3-5 Mb 10-15% Yes Yes Yes Yes chromosomes (550 bands) Metaphase FISH 100’s kb n/a Yes No Yes Yes Interphase FISH 100’s kb 1-5% No No Yes Yes GMA 10-100’s kb 10-20% No Yes Yes No  Sizes: kb=1x103, Mb=1x106  Structural abnormalities Deletions Duplications Insertions Terminal Interstitial Reciprocal Translocations Robertsonian Translocations Balanced Unbalanced Balanced Unbalanced  Incidence of chromosome abnormalities detected in newborns Abnormality Rate/1000 Rate (1/n) Autosomal Trisomy 1.62 617 Sex Chromosome Aneuploidies (All) 2.70 375 Balanced Structural Rearrangements 2.04 490 Translocations, insertions 0.97 1,028 Inversions 0.16 6,331 Robertsonians 0.91 1,099 Unbalanced Structural Rearrangements 0.63 1,587 Translocations, insertions, inversions 0.09 10,935 Robertsonians 0.07 13,366 Deletions, rings 0.06 17,184 +Markers (e.g. isochromosomes) 0.41 2,455 Data 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 a congenital anomaly syndrome – Most risk figures fall into the range of 0-30% for a liveborn child with an abnormality (higher end if previous child) May disrupt gene expression (breakpoint within a gene or regulatory element by position effect) – In prenatal setting and de novo, risk ~6% (Warburton ‘91) Create gene fusions and affect gene expression by position effect – Esp. in cancer ex. t(9;22) BCR-ABL1 chimeric transcript or t(11;14) CCND1 upregulation by translocation near the IGH locus regulatory region Pachytene configuration (quadrivalent) in the balanced translocation carrier/translocation heterozygote A, B: Normal chromosomes A’, B’: Derivative chromosomes Gardner, Sutherland and Shaffer. 2012  Modes of Segregation During Gametogenesis in the Balanced Translocation Carrier Only 2:2 alternate segregation will result in normal/balanced gametes All other modes of segregation result in unbalanced gametes Chromosome Abnormalities and Genetic Counseling. 4th ed. Gardner, Sutherland and Shaffer. 2012  Predicting clinical outcomes for the balanced translocation carrier Factors that influence segregation and outcomes Location of the breakpoints, relative to chromosome size and the centromere Relative size of chromosomes involved  See also Table 5-4 in Gardner, Sutherland and Shaffer 2012 Gardner, Sutherland and Shaffer. 2012  Tertiary trisomy in the t(11;22)(q23;q11) carrier 46,t(11;22) 47,+der(22),t(11;22) (Emanuel syndrome) Tertiary trisomy 3:1 segregation Gardner, Sutherland and Shaffer. 2012  Predicting clinical outcomes for the balanced translocation carrier Factors 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 Table 5-4 in Gardner, Sutherland and Shaffer 2012 Gardner, Sutherland and Shaffer. 2012  Pedigree of a family carrying a translocation with a large centric segment Gardner, Sutherland and Shaffer. 2012  Structural abnormalities Deletions Duplications Insertions Terminal Interstitial Reciprocal Translocations Robertsonian Translocations Balanced Unbalanced Balanced Unbalanced  Robertsonian translocations Frequency ~1/1000, 95% are nonhomologous – rob(13;14) is most common (1:1300) Homology and orientation of sequences in p-arm stalks of chrs 13, 14 and 21 likely explain relative prevalence of rob(13;14) and rob(14;21) amongst carriers (via NAHR) Gardner, Sutherland and Shaffer. 2012  Robertsonian translocations: Meiotic segregation Trivalent Balanced Unbalanced Gametes Normal Carrier Trisomy Monosomy Modified from Gardner, Sutherland and Shaffer. 2012  Imprinted chromosomes and human disease due to uniparental disomy (UPD) Image from: http://carolguze.com/text/442-10- nontraditional_inheritance.shtml Velissariou, Balkan J Med Gen  Risk for uniparental disomy (UPD) Risk for expression of clinical phenotype if rob chromosome contains imprinting genes (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 imprinting genes (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 Robertsonian translocation carrier Risk to have unbalanced is greater for females 10-15% for chromosomes 21 Risk for UPD is the same The risk to homologous rob carriers is ~100% Very rare instances of post-zygotic correction are reported Gardner, Sutherland and Shaffer. 2012  Modes of X-inactivation Morey 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 cell selection (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 of X-chromosome abnormalities in females  Most structural abnormalities and some mutations lead to non-random inactivation Key Active X = White Inactive X = Gray * * = XIST Leppig and Disteche, Semin Reprod Med, 2001  Translocation X;A in females-balanced carriers may also be affected, dependent on X-inactivation Key There is an inherent risk to the balanced Active X = White female carrier if X inactivation is not Inactive X = Gray skewed to preferentially inactivate the Autosomal material = hashed normal X * = XIST Risk for functional disomy (double expression of X-linked genes relative to their normal level) of the translocated X segment on the der(A) Risk for functional monosomy of the translocated autosomal segment on the der(X) Leppig and Disteche, Semin Reprod Med, 2001  Structural abnormalities Inversions Ring chromosomes Pericentric Paracentric inversion inversion Recombinant Isochromosomes chromosomes  Recombinant chromosome arises from a parental pericentric inversion 1 1 2 6 3 5 4 4 3 5 2 6 7 7 7 1 6 2 5 3 4 4 3 5 6 2 7 7 Image source: http://www.ucl.ac.uk/~ucbhjow/bmsi/bmsi_7.html rec(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 post- bone marrow transplant (BMT)  Basic terminology for classifying hematologic malignancies Leukemia: cancer of the blood and/or bone marrow Lymphoma: cancer in the lymphatic tissue (nodal or extranodal) Myeloid: cells that arise and differentiate in the bone marrow (RBC’s, platelets, WBCs: granulocytes) Lymphoid: cells that arise in the bone marrow and differentiate and/or function in the lymphatic system (WBC types: B-cells, T-cells, NK cells)  Blood Cell Lineages Myeloid-type Lymphoid-type diseases diseases AML ALL CML CLL MDS MM MPD Lymphomas Image source: http://www.allthingsstemcell.com/wp-content/uploads/2009/02/hematopoiesis_simple1.png  Types of Chromosome Abnormalities in Cancer Numerical – Aneuploid: 2n - or + chromosomes Monosomy or trisomy – Polyploid: 1n, 2n, 3n, 4n, etc. where n=23 chr. Structural – Deletions – Duplications/amplifications – Translocations: balanced or unbalanced – Inversions Copy-neutral loss of heterozygosity (LOH) – Mitotic recombination – Mitotic malsegregation: uniparental disomy  Comparing technologies… Aberrations of copy number, structure Aberrations of genotype Balanced Unbalanced Karyotyping + + + + +/- + - - - FISH + + + + + + + - - CMA (SNP) + + - + + + + + + Image modified from Albertson et al., 2003, Nature Genetics  Defining clonality/acquired changes in oncology studies Karyotyping: – At least two metaphase cells with the same extra chromosome, structural abnormality – At least three metaphase cells with the same chromosome loss FISH: – Abnormality observed in a percentage of cells (usually >1-5%), 200 interphase FISH cells are examined Genomic microarray: – Evidence of mosaicism in the sample as shown by the copy number and/or SNP-containing probes – Cannot determine whether multiple mosaic abnormalities represent different clones/evolution (clonal diversity) Karyotyping in Cancer e.g. Clinical Utility of Karyotype in ALL Cytogenetic subtype distribution by age Proportion of cases Harrison. ASH Education Program (2013) 118-125 The Genetic Basis of Cancer Types of genes involved in cancer Calvert and Frucht, 2002, Ann Int Med  Types of genes in cancer Oncogenes: mutant forms of genes (proto- oncogenes) that positively regulate cell proliferation and survival Dominant, gain-of-function type mutations Image source: http://www.scq.ubc.ca/images/oncogeneformation.gif  Mechanisms of oncogene activation Chromosomal rearrangements (translocations, inversions) – A gene fusion creating a chimeric protein – Upregulation of gene expression by position effect Copy number gains – Trisomy, tetrasomy, etc. – Gene amplification Image modified from Albertson et al., 2003, Nature Genetics Oncogene Activation by Gene Fusion  t(9;22) in chronic myelogenous leukemia (CML) First chromosomal abnormality associated with cancer, discovered in 1960 Abnormal Chr. 22 named the Ph Philadelphia (Ph) chromosome Image source: http://atlasgeneticsoncology.org/Anomalies/t0922CML.html der(9) 9 22 der(22) The t(9;22)(q34;q11) reciprocal translocation (Proto-oncogene)  BCR/ABL1 protein is a constitutively active tyrosine kinase The N-terminal cap regulates controlled ABL kinase activity Fusion to 5’ BCR – Increases cell proliferation – Inhibits programmed cell death – Increases invasiveness – Inhibits DNA repair Goldman and Melo, NEJM, 2003  Targeted Therapy: Inhibitors of tyrosine kinase (TKIs) Imatinib mesylate (Gleevec) BCR-ABL1 kinase inhibited by Imatinib was the first TKI approved by the FDA in 2001 Mechanism: Competes with ATP for binding sites Inhibits progression of CML in the majority of patients Drug resistance can develop over time Image source: http://upload.wikimedia.org/wikipedia/commons/c/ca/Bcr _abl_STI_1IEP.png Oncogene Activation by Position Effect  c-MYC rearrangements in Burkitt lymphoma Cell of origin is a peripheral memory B-cell c-MYC at 8q24 is a proto- IGΚ locus on 2p oncogene is a transcription factor that induces cell proliferation Immunoglobulin genes are strongly expressed in B-cells Translocation juxtaposes c-MYC IGH locus on 14q with IG enhancers t(8;14)(q24;q32) in 75-85% cases t(8;22)(q24;q11) in ~10% cases IGL locus on 22q t(2;8)(p12;q24) in ~5% cases Image source: http://atlasgeneticsoncology.org/Anomalies/t0814ID1050.h tml C-Myc influences the transcription of a variety of proteins involved in the cell cycle Blum et al., Blood. 2004  Selected Rearrangements in Cancer Neoplasm Translocation Percentage of Oncogene Cases Chronic myelogenous t(9;22)(q34;q11) 100% (includes BCR-ABL1 leukemia variant fusions) Acute lymphocytic t(9;22)(q34;q11) 10-15% BCR-ABL1 leukemia Acute lymphocytic t(4;11)(q21;q23) 5-10%; 40%