Cytogenetics: Nomenclature and Disease

Cytogenetics: Nomenclature and Disease

Willis Navarro, MDMedical Director, Transplant Services

National Marrow Donor Program

OverviewOverview

• Normal ChromosomesNormal Chromosomes– Structure

Genes– Genes• Chromosomal Disruptions

f C C– Types of Chromosomal Changes• Disruptions 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

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

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

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

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

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

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

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

Monosomy X: Turner Syndrome C i i l LConstitutional Loss

Trisomy 21: Down SyndromeC i i l G iConstitutional Gain

DeletionDeletion

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

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

InversionsInversions

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

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

TranslocationsTranslocations

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

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

Dicentric ChromosomeDicentric Chromosome

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

IsochromosomesIsochromosomes

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

Ring ChromosomesRing Chromosomes

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

DuplicationDuplication

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

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

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]

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

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

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)

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)

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

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”

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

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.

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

Web ReferencesWeb References

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

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