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Cytogenetics: Nomenclature and Disease Willis Navarro, MD Medical Director, Transplant Services National Marrow Donor Program

Cytogenetics: Nomenclature and Disease

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Page 1: Cytogenetics: Nomenclature and Disease

Cytogenetics: Nomenclature and Disease

Willis Navarro, MDMedical Director, Transplant Services

National Marrow Donor Program

Page 2: Cytogenetics: Nomenclature and Disease

OverviewOverview

• Normal ChromosomesNormal Chromosomes– Structure

Genes– Genes• Chromosomal Disruptions

f C C– Types of Chromosomal Changes• Disruptions and Disease

Page 3: Cytogenetics: Nomenclature and Disease

Structural OverviewStructural Overview• DNA forms a double

helixhelix• Double helix

structure is wound around histones

• DNA/histone complex th f ththen forms the chromosome structure

http://ghr.nlm.nih.gov/handbook/illustrations/chromosomestructure

Page 4: Cytogenetics: Nomenclature and Disease

Cell Division and CytogeneticsCell Division and Cytogenetics• Tissue cells of interest

are grown in culture• Cell must be “frozen” at

t hmetaphase– Mitotic inhibitor added– Chromosomes condensed– Cells harvested

From: http://www.google.com/imgres?imgurl=http://nte-serveur.univ-lyon1.fr/nte/EMBRYON/www.uoguelph.ca/zoology/devobio/miller/mitosis1.gif&imgrefurl=http://nte-serveur.univ-lyon1.fr/nte/EMBRYON/www.uoguelph.ca/zoology/devobio/210labs/mitosis1.html&h=538&w=450&sz=24&tbnid=PXVEm8paeVwJ::&tbnh=132&tbnw=110&prev=/images%3Fq%3Dmitosis&hl=en&usg=__XTdVA_DgEhY6qk-d9HDMvHcVN9Q=&sa=X&oi=image_result&resnum=4&ct=image&cd=1

Page 5: Cytogenetics: Nomenclature and Disease

Human Chromosome BasicsHuman Chromosome BasicsC

metacentric

C

metacentric

submetacentric

C

C

C

Acrocentric

From: http://www.miscarriage.com.au/images/pages/karyotype_normal.jpg

• 22 pairs plus 2 sex chromosomes (diploid number: 46): (46, XX)• Composed of DNA plus infrastructure (histones, proteins, RNA, sugars)• 3 groups of shapes based on centromere position, arm length

Page 6: Cytogenetics: Nomenclature and Disease

Example of G-Banding: Ch 11Chromosome 11

• GTL stain:• GTL stain: Giemsa/Trypsin/Leishman

• Chr 11 is submetacentricR t ti id

p arm

• Representative ideogram• Stained to distinguish denser

and less dense areasCentromere

• Unique staining patterns for each chromosome

• Many genes codedq arm

• Banding ≠ genes

From: http://upload.wikimedia.org/wikipedia/commons/thumb/c/cf/Chromosome_11.svg/164px-Chromosome_11.svg.png

Page 7: Cytogenetics: Nomenclature and Disease

How Do You Define a Gene?How Do You Define a Gene?• DNA sequence q

begins with a start codon; ends with a stop codonp

• Amino acids (each with a 3-character code) then join tocode) then join to form a protein which then has a functionTh i “fill ” DNA• There is “filler” DNA that codes for other stuff

Page 8: Cytogenetics: Nomenclature and Disease

So Many GenesSo Many Genes…• ARHGAP20 109952.97611q23.2ARHGEF12 119713.15611q23.3ATM 107598.76911q22-q23BAD

63793.87811q13.1BIRC3 101693.40411q22C11orf30 75833.71711q13.5CARS 2978.73511p15.5CASP1104401.44711q23CBL 118582.20011q23.3-qterCCND1 69165.05411q13CHKA 67576.90211q13.1DDB247193.06911p12-p11DDX10 108041.02611q22-q23EXT2 44073.67511p12-p11FANCF 22600.65511p15HRAS522.24211p15.5KAT5 65236.06511q13LMO2 33836.69911p13MAML2 95351.08811q21MEN164327 57011q13MIRN125B1 121475 67511MLL 117812 41511q23MRE11A 93790 11511q21MYEOVp arm 64327.57011q13MIRN125B1 121475.67511MLL 117812.41511q23MRE11A 93790.11511q21MYEOV68818.19811q13.2NUMA1 71391.55911q13NUP98 3689.63511p15PAK1 76710.70811q13-q14PICALM85346.13311q14POU2AF1 110728.19011q23.1RELA 65178.39311q13RSF1 77054.92211q13.5SDHD111462.83211q23SPA17 124048.95011q24.2SPI1 47332.98511p12-p11.22WT1 32365.90111p13ZBTB16113436.49811q23 ACAT1 107497.46811q22.3ACP2 47217.42911p11.2ADAMTS15129824.07911q25ADAMTS8 129780.02811q25ADM 10283.21811ADRBK1 66790.48111q13AHNAK62039.95011q12-q13AIP 67007.09711q13.3ALDH3B2 67186.20911q13ALKBH3 43858.97311p11.2ALKBH8106880.54411q22.3AMPD3 10428.80011p15ANKK1 112763.72311q23.2ANO1 69602.05611q13.2APBB16372.93111p15API5 43290.10911APLNR 56757.64311q12APOA1 116211.67911q23-q24ARAP172073.76211q13.3ARCN1 117948.32111q23.3ARFIP2 6453.50211p15ARHGAP146655 20811p11 2ARHGEF17 72697 31711q13 3ARL2 64538 16211q13ARRB1 74654 13011q13ART1

Centromere

p arm

46655.20811p11.2ARHGEF17 72697.31711q13.3ARL2 64538.16211q13ARRB1 74654.13011q13ART13622.93711p15ASCL2 2246.30411p15.5B3GAT1 133753.60811q25B3GNT6 76423.08311q13.4BARX2128751.09111q24.3BCL9L 118272.06111q23.3BDNF 27633.01811p14.1BIRC2 101723.17611q22BRMS165861.38011q13-q13.2BRSK2 1367.70511p15.5BTG4 110843.46611q23C11orf17 8889.27711p15.3C11orf6865440.85911q13.1C1QTNF4 47567.79211q11C1QTNF5 118714.86211q23.3CADM1114549.55511q23.2CALCA 14946.62411p15.2CAPN1 64705.91911q13CASP4 104318.80411q22.2-q22.3CASP5 104370.17211q22.2-q22.3CAT 34417.05411p13CCDC73 32580.20211p13CCKBR6237.54211p15.4CD151 822.95211p15.5CD248 65838.53411q13CD3E 117680.50511q23CD4435116.99311p13CD5 60626.50611q13CD59 33681.13211p13CD6 60495.69111q12.2CD81 2355.12311pter-p11.2CD82 44543.71711p11.2CDC42EP2 64838.90711q13CDK2AP2 67030.54411q13CDKN1C2861 02411p15 5CDON 125331 92311q23-q24CENTD2 72073 76211q13 3CEP57 95163 29011q21CFL1q arm 2861.02411p15.5CDON 125331.92311q23 q24CENTD2 72073.76211q13.3CEP57 95163.29011q21CFL165378.86111q13.1CHEK1 125000.24611q24.2CHORDC1 89574.26511q14.3CHRDL2 74085.12211CKAP546721.66011p11.2CLCF1 66888.21511q13.3CLP1 57181.20611q12CNTF 58146.72111q12CNTN598397.08111q21-q22.2COP1 104417.263-COX8A 63498.65511q12-q13CPT1A 68278.66411q13.2CREB3L146255.80411q11CRTAM 122214.46511q24.1CRY2 45825.24511p11.2CRYAB 111284.56011q22.3-q23.1CST665536.03811q13CTNND1 57285.81011q12.1CTSD 1730.56111p15.5CTSF 66087.51111q13.2CTTN69922.26011q13CUL5 107384.61811q22.3CXCR5 118259.75111q23.3CYB5R2 7642.90211p15.4DCPS125678.85711q24DDB1 60823.49511q12-q13DDX25 125279.48211q24DDX6 118123.68311q23.3DEAF1634.22511p15.5DGKZ 46339.72111p11.2DKK3 11941.11911p15.3DLAT 111400.74811q23.1DNAJC463754.32911q13DPF2 64857.92211q13.1DPP3 66004.45611q12-q13.1DRD2 112785.52711q23DUSP81531.85711p15.5DYNC2H1 102485.37011q21-q22.1EED 85633.46311q14.2-q22.3EHF 34599.24411p12EI24

q arm

1531.85711p15.5DYNC2H1 102485.37011q21 q22.1EED 85633.46311q14.2 q22.3EHF 34599.24411p12EI24124944.50811q23EIF3F 7965.44311p15.4EIF4G2 10775.16911p15ELF5 34456.91811p13-p12ESRRA63829.62011q12ETS1 127833.87011q23.3F2 46697.31911p11-q12FADD 69726.91711q13.3FADS261352.28911q12.2FAM111B 58631.28611q12.1FAM89B 65096.39611q23FAT3 91724.91011q14.3FAU64644.67811q13FBXL11 66644.07411q13.1FCHSD2 72225.43811q13.3FEN1 61316.72611q12FEZ1124820.85811q24.2FGF19 69222.18711q13.1FGF3 69333.91711q13FGF4 69296.97811q13.3FLI1128069.02311q24.1-q24.3FLRT1 63627.93811q12-q13FOLH1 49124.76311p11.2FOLR1 71578.60711q13.3-q14.1FOLR2 71605.46711q13.3-q14.1FOSL1 65416.26811q13FOXR1 118347.62711q23.3FRAG13786.36011p15.5FSHB 30209.13911p13FTH1 61488.33311q13FUT4 93916.77511q12-qterFXC16459.25311p15.2-q15.5FZD4 86334.36911q14-q21GAB2 77603.99011q14.1GAL 68208.55911q13.2GAS222652.93111p14.3GCRG224 82211.05611q13-q14GNG3 62231.70911p11GRM5 87880.62611q14.3GSTP1

Dessen P, Knuutila S, Huret JL Chromosome 11. Atlas Genet Cytogenet Oncol Haematol. 2002. 22652.93111p14.3GCRG224 82211.05611q13 q14GNG3 62231.70911p11GRM5 87880.62611q14.3GSTP1

67107.64211q13-qterGTF2H1 18300.71911p15.1-p14H19 1972.98311p15.5H2AFX 118469.79511q23.3HBB5203.27211p15.5HCCA2 1447.26911p15.5HEPACAM 124294.35611q24.2HEPN1 124294.356-HIPK333235.74411p13HPS5 18256.79311p14HRASLS2 63076.81811q12.2HRASLS3 63098.52011q12.3HSPA8122433.41011q24.1HSPB2 111288.70911q22-q23HTATIP2 20341.86511HTR3A 113351.12011q23.1-q23.2HYOU1 118420.10611q23.1-q23.3ICEBERG 104513.87911q21-q22IFITM1 303.99111p15.5IFITM3309.67311p15.5IGF2 2106.92311p15.5IGF2AS 2118.31311p15.5IGHMBP2 68427.89511q13.3IL10RA117362.31911q23IL18 111519.18611q22.2-q22.3ILK 1132.47411p15.5MUC5B 1200.87211p15.5MUC61002.82411p15.5MUS81 65384.44811q13MYOD1 17697.68611p15NAALAD2 89507.46611q14.3-q21NAP1L4…

URL : http://AtlasGeneticsOncology.org/Indexbychrom/idxa_11.html © Atlas of Genetics and Cytogenetics in Oncology and Haematology

Page 9: Cytogenetics: Nomenclature and Disease

What can go wrong with a gene?What can go wrong with a gene?

• The correct sequence is critical to codingThe correct sequence is critical to coding the right protein/protein structure

• If the chromosome carrying a particular• If the chromosome carrying a particular gene is altered, then the resulting mutated protein or control elements may causeprotein or control elements may cause problems

Page 10: Cytogenetics: Nomenclature and Disease

What can go wrong with a h ?chromosome?

• Constitutional vs acquired abnormalitiesq

• Numerical abnormalities– Monosomy: loss of a whole chromosome– Trisomy: gain of a whole chromosome

• Structural abnormalities– Deletions– Deletions– Inversions– Translocations

Page 11: Cytogenetics: Nomenclature and Disease

Monosomy X: Turner Syndrome C i i l LConstitutional Loss

Page 12: Cytogenetics: Nomenclature and Disease

Trisomy 21: Down SyndromeC i i l G iConstitutional Gain

Page 13: Cytogenetics: Nomenclature and Disease

DeletionDeletion

http://ghr.nlm.nih.gov/handbook/illustrations

Page 14: Cytogenetics: Nomenclature and Disease

Deletion 5qA i d LAcquired Loss

• Interstitial losses of the long arm of chromosome 5Th l l i• These losses result in large numbers of genes being lostgenes being lost

• Often associated with myelodysplastic syndromes and acute myeloid leukemia From: http://atlasgeneticsoncology.org/Educ/Images/GeneticCancerFig7.jpg

Page 15: Cytogenetics: Nomenclature and Disease

InversionsInversions

http://ghr.nlm.nih.gov/handbook/illustrations

Page 16: Cytogenetics: Nomenclature and Disease

Inversion (3)(q24q27)A i d Ab liAcquired Abnormality

I t titi l t i tFrom: http://members.aol.com/chrominfo/images/inv3ideo.gif

• Interstitial segment inverts

Page 17: Cytogenetics: Nomenclature and Disease

TranslocationsTranslocations

http://ghr.nlm.nih.gov/handbook/illustrations

Page 18: Cytogenetics: Nomenclature and Disease

Translocation t(9;22)A i d Ab liAcquired Abnormality

• Material isMaterial is exchanged between chromosomes 9 and 22 creating a new22, creating a new fusion gene: bcr/abl

• Breakpoint may vary a bit such that thea bit such that the newly created fusion protein may be of several lengthsseveral lengths– p190 (kDa)– p210 From: http://members.aol.com/chrominfo/images/inv3ideo.gif

http://atlasgeneticsoncology.org/Anomalies/CML.html

Page 19: Cytogenetics: Nomenclature and Disease

Dicentric ChromosomeDicentric Chromosome

http://ghr.nlm.nih.gov/handbook/illustrations

Page 20: Cytogenetics: Nomenclature and Disease

IsochromosomesIsochromosomes

http://ghr.nlm.nih.gov/handbook/illustrations

Page 21: Cytogenetics: Nomenclature and Disease

Ring ChromosomesRing Chromosomes

http://ghr.nlm.nih.gov/handbook/illustrations

Page 22: Cytogenetics: Nomenclature and Disease

DuplicationDuplication

http://ghr.nlm.nih.gov/handbook/illustrations

Page 23: Cytogenetics: Nomenclature and Disease

Recap of Basic AbnormalitiesRecap of Basic Abnormalities• Loss or gain of entire chromosomes

Monosomy– Monosomy– Trisomy

• Structural– Deletions

I i– Inversions– Translocations

• Plus more uncommon types of abnormalities– Derivative chromosome (der)

• Used when only one chromosome of a translocation is present or• One chromosome has two or more structural abnormalities

– Dicentric chromosome (dic) [chromosome has two centromeres]– Duplicate (dup) [duplication of a portion of a chromosome]

Insertion (ins)– Insertion (ins)– Isochromosomes (i) [both arms are the same]– Marker chromosome (mar) [unidentifiable piece of chromosome]– Ring chromosome (r)

Hyperdiploidy: greater than 48 chromosomes– Hyperdiploidy: greater than 48 chromosomes

Page 24: Cytogenetics: Nomenclature and Disease

Interpreting Cytogenetic ReportingInterpreting Cytogenetic Reporting

• In sequence:q– the overall number of chromosomes identified– sex chromosomes– affected chromosomes– type of abnormalities described in shorthand– chromosomal band location– In brackets, the number of cells with a given karyotypeIn brackets, the number of cells with a given karyotype

• Examples– 46, XX; t(9;22)(q13;q22) [20]

47 XY; +21 [12]– 47, XY; +21 [12]– 46, XX; inv 16(q13; q21) [20]– 45, XY; -5 [18]; 46, XY [2]

46 XY 5 (q13) [4] 46 XX [16]– 46, XY; -5 (q13) [4]; 46, XX [16]

Page 25: Cytogenetics: Nomenclature and Disease

Cytogenetic PioneersCytogenetic PioneersBarbara McClintock

Fi t ti f i– First genetic map of maize– Genetic and physical characteristics correlated– Her work helped explain how cells that share the

h diff t f tisame genome can have different functions– Nobel Prize for transposons in 1983

Janet Rowley– Hypothesized that leukemias might

contain non-random geneticcontain non random genetic abnormalities

– 1972: Showed that recurring chromosomal abnormalities occurred in leukemia and sometimes defined the disease’s characteristics

Page 26: Cytogenetics: Nomenclature and Disease

Leukemias and CytogeneticsLeukemias and Cytogenetics• Certain morphologic subtypes were

known to have distinct prognosesknown to have distinct prognoses and/or clinical syndromes (M0-M7)

• Examples:– acute promyelocytic leukemia (M3)p y y ( )

• High bleeding risk due to coagulopathy but favorable prognosis

• t(15; 17)(q22;q12)– AML, subtype M4eo

• Favorable prognosis• inv(16)(p13;q22)

– Chronic myeloid leukemia• t(9;22)(q11;q34)( ; )(q ;q )

– Some myelodysplastic patients—typically older women--had a pattern of normal platelet counts and a favorable prognosis

• 5q- syndrome

Page 27: Cytogenetics: Nomenclature and Disease

Risk Stratification for Acute L k i U i C iLeukemias Using Cytogenetics

• Previous to Janet Rowley and others’ observations about cytogenetics and prognosis, leukemias were only categorized by morphology under the microscopeleukemias were only categorized by morphology under the microscope

• AML– Favorable Risk

• inv (16)• t (8;21)• t (15;17)

– Intermediate Risk• All abnormalities not in favorable or high risk categories, plus normal

– Poor Risk• Monosomy 5 or 7; 5q-; 7q-• t (9;22)• Complex (3 or more abnormalities)

• ALL– Favorable risk

• Hyperdiploidy– High Risk

• t (1;19)• t (9;22)• t (4;11)

Page 28: Cytogenetics: Nomenclature and Disease

Pre-TED Form (1): Cyto dataPre TED Form (1): Cyto data

• First three AML cyto abnormalities are associated with favorable prognosis (AML/ETO, e.g., refers to the two genes involved in the leukemia)

• AML with 11q23: often associated with previous topoisomerase inhib.-based chemotherapy (MLLtopoisomerase inhib. based chemotherapy (MLL gene is located at 11q23); usually t(9;11) (p22;q23)

Page 29: Cytogenetics: Nomenclature and Disease

Pre-TED Form (2)Pre TED Form (2)

I th TED l b Ph f t• In the pre-TED example above, Ph+ refers to the cytogenetics; bcr refers to the detection of bcr/abl gene product usually by PCR or FISHbcr/abl gene product, usually by PCR or FISH

Page 30: Cytogenetics: Nomenclature and Disease

Form 2100 Chimerism StudiesForm 2100 Chimerism Studies

N t th t f ll ti f• Note that for collection of chimerism data, PCR is not an option and should not beoption and should not be recorded under “other”

Page 31: Cytogenetics: Nomenclature and Disease

Disease Status: FISHDisease Status: FISH

• For data purposes, FISH is a subset of cytogeneticsa subset of cytogenetics (cellular level)

• Molecular evidence would be PCR and similarbe PCR and similar

Page 32: Cytogenetics: Nomenclature and Disease

The Future of Prognosticating Outcomes in Acute Leukemia

• May be based on the molecular biology of theMay be based on the molecular biology of the leukemia as ascertained by– PCR– FISH– Microarray data/gene profiling

• More and more critical to understand the molecular basis as more targeted therapies b il blbecome available– Anti-bcr/abl drugs: imatinib and 2nd generation drugs

Anti flt3 etc– Anti flt3 etc.

Page 33: Cytogenetics: Nomenclature and Disease

SummarySummary• A variety of chromosomal abnormalities can be y

characterized and described using cytogenetics• Non-random chromosomal alterations occur, can

define the disease (e.g. APML), and can havedefine the disease (e.g. APML), and can have important prognostic value

• Not all genetic abnormalities can be seen using cytogenetic techniques (e g normal cytogeneticscytogenetic techniques (e.g. normal cytogenetics in AML)

• Newer techniques (polymerase chain reaction [PCR] fl t i it h b idi ti [FISH])[PCR], fluorescent in-situ hybridization [FISH]) can assist in searching for occult genetic aberrations

Page 34: Cytogenetics: Nomenclature and Disease

Web ReferencesWeb References

• http://www.slh.wisc.edu/wps/wcm/connect/et t/ t ti /xtranet/cytogenetics/

• http://ghr.nlm.nih.gov/handbook/illustrations