Cytogenetic abnormalities

Presenter -Dr Dhanya A NModerator–Dr chaithra

Contents • Introduction to cytogenetics• Milestones in cytogenetics • Chromosome • Cell cycle and cell division• Indications for cytogenetic analysis • Approach to cytogenetic analysis • Chromosomal abnormalities chromosomal disorders chromosomal abnormalities in neoplasm - hematological - soft tissue tumors

Introduction to cytogenetic

CYTOGENETICS DEFINITION :Cytogenetics is the study of chromosomes and its abnormalities: alteration in the number and structure.

MILESTONES IN CYTOGENETICS:

• Arnold: First observed chromosomes:1879• Hansemann & Flemming Counted the chromosomes:

1891• Winiwarter: Isolated X chromosome• Painter: Isolated Y chromosome• Tijio and Levan: 1956: Described correct

chromosome number as 22 pair of autosomes and 2 sex chromosomes.

• Levan introduced the use of Colchicine to arrest mitosis at metaphase.

• Hsu,Makino & Nishimura and Hughes- Hypotonic technique: In 1952 in karyotyping.

• Gall and Prudue described in situ hybridization techniques.

Chromosome

Is a packaged and organized structure containing the DNA of a living organism

Types of chromosomes

CELL CYCLE

• Interphase – G1, S, G2

• M phase (Mitosis)

• G0 – NO cell division

INDICATIONS FOR CYTOGENETIC ANALYSIS

• Prenatal – pregnancies involving older (>35yrs) women.

• Confirmation or exclusion of diagnosis - known chromosomal syndromes.

• Unexplained psychomotor retardation with or without dysmorphic features.

• Abnormalities of sexual differentiation and development - ambiguous genitalia.

Continued..

• Recurrent miscarriage, stillbirth or spontaneous abortions.

• Females with proportionate short stature and primary amenorrhea.

• Parents and children of persons with chromosomal translocations, deletions and duplications.

• Pregnancies at risk of aneuploidy from results of fetal ultrasound.

• Neoplastic conditions- soft tissues and hematological.

APPROACH TO THE DIAGNOSIS OF CYTOGENETIC DISORDERS

1. Karyotyping2. Insitu hybridization3. Fluorescence insitu hybridization4. Spectral karyotyping5. Comparative genomic hybridization

KARYOTYPE

• Standard display of stained and photographed chrosmosomes in metaphase spread, arranged in pairs, in order of decreasing length.

• Human somatic cells - 22 pair of autosomes identical in male and female

• 2 sex chromosomes XX - female & XY - males.

1. TISSUE SAMPLES & CELL CULTURE:

• Prenatal- Amniotic fluid - 20ml - Chorionic villi- 25mg of Vascularised and budding

villi from chorion frondosum - PUBS(percutaneous umbilical blood sampling)

• Postnatal – Peripheral blood – 4 ml of heparinised – Skin fibroblasts- 4mm diameter– Bone marrow- 1 ml of heparinised bone

marrow.– Lymph node- 0.5 to 1 cm3– Solid tumors- Part of specimen submitted for

histopathological examination. Ideally 0.5-1 gm

2. Culture• Culture medium – Preservative free sodium

heparin, (RPMI 1640 ), mitotic stimulant (phytohemagglutinin) and antibiotics( penicillin, streptomycin ).

• Short term culture: 1-3 days – blood, bonemarrow, chorionic villi

• Long term culture: 1-3 wks - other tissue types

3. Arrest of cell division: at metaphase, by Colchicine [Deacetyl methyl colchicine] for 20 min.

4. Cells harvested – centrifugation Incubated for 10min in hypotonic solution (dilute solution of KCl 0.075 mol) .

5. Cell fixation- 3:1 methanol/ glacial acetic acid mixture (carnoy’s) for 30min.

6. Staining : Trypsinization of the chromosomes prior to

staining, weakens the DNA-Protein interactions, add buffer (Na2HPO4 and NaH2PO4), banding techniques done with the dyes.

7. Microscopic analysis and photography

8. Karyotype production (manual/automated)

9. Interpretation

Staining

Q-banding-• The first banding method

developed• Uses quinacrine mustard or

quinacrine dihydrochloride,• creates a flourescent

transfers band on exposure to UV light,

• Q- bands fade over time not routinely used .

G-banding• Uses Giemsa dye to produce

transverse bands light (G-C rich DNA) dark band (A-T rich DNA)

• G-bands are identical to Q-bands

• G-banding is the most widely used banding technique for routine chromosome analysis

• Around 400 bands per haploid genome seen. Each band corresponds to 5-10megabases

R-banding• Treating chromosomes with a

hot alkaline solution before Giemsa staining

• Produces bands that are the reverse of G-bands, called R-bands.

• In R banding telomeres should appear as dark bands and their absence as the result of deletion is more obvious.

C-banding• selectively stains constitutive

heterochromatin, and are located at all centromeres and distal long arm of Y chromosome.

• Staining with giemsa followed by heat denaturation results in darkly staining heterochromatic regions at centromere with light staining chromosome arms.

Nuclear organizing region(NOR) banding • Specific chromosomal region that forms and

maintain the nucleoli are called NOR.• NOR located on stalk of acrocentric chromosomes

and contain gene for 18S and 28S rRNA.• Stained by Geimsa (N- banding) or silver

impregnation ( Ag-NOR)

Successful cytogenetic analysis depends on – - Cells must be in adequate numbers - Analysis must be performed on viable cells in division - Chromosomes must be separated from one another - Chromosomes must be identified & characterized normal/abnormal - Arranged according to the length in a decreasing order.

• Karyotyping Chromosome from each metaphase spread are arranged in prescribed order – karyotype cells – imaged, printed & karyotyped

ISCN International System for Human Cytogenetic Nomenclature.• Centromere divide the chromosome into short arm and long am • Chromosome arms divided into regions on the basis of landmarks The region adjacent to centromere of short arm and long arm

are given number 1 as p1, q1 respectively, the next distal region is given 2 and so on

• The regions are subdivided into bands and the bands are subdivided into sub bands as the resolution increases and the numbering done sequentially

ISCN of chromosome 11

In situ hybridization

• Hybridization refers to the binding or annealing of complementary DNA or RNA sequences

• Main purpose – detection of specific nucleic acid sequences in chromosomes.

• In early studies, radio isotopes were used as labels for nucleic acids, and detection of hybridized sequence were done with autoradiography.

• As technology advanced, detection by enzymatic and fluorescent means become available for quick and safe analysis.

• Uses- Detection of missing,additional chromosomes,chromosome rearrangements and microdeletions.

Fluorescent in situ hybridization

The probe and metaphase target are denatured by a high

temperature and formamide.

Probe is hybridized to the chromosomal target.

Unbound probe is removed by post hybridization washes.

Bound probe is detected by fluorescence microscopy

Types of probes 1] Centromere enumerating

probe(CEP) - Bind to highly repitative sequence

alpha satellite sequences of centromere and produce strong signals.

Similar sequences in pericentric region results in cross hybridization artifact.

2] Locus specific identifier(LSI) probe

Target distinct chromosomal region of interest and utilize single copy rather than repetitive DNA.

3] Whole chromosomes probes Also known as chromosome

painting probes or chromosomes libraries, consists of thousands of overlapping probes that recognize unique and moderately repetitive sequences along the entire length of individual chromosomes

Advantages – - Many more cells can be examined at a single time.- Metaphases are not essential, so abnormalities can

be detected in non dividing cells.- Can be performed in a shorter period of time.- Abnormalities that cannot be detected by

conventional cytogenetic analysis may be detected.Main disadvantage – - Only those abnormalities that are specifically sought

will be found whereas conventional analysis permits all chromosomes to be evaluated

SPECTRAL KARYOTYPING (multicolor fluorescence in-situ hybridization)

• 24-colour, multi-chromosomal painting assay that allows visualization of all human chromosomes in one experiment.

• Uses –1. Ability to detect complex chromosomal

rearrangements.2. Identifies marker chromosomes– makes this highly

sensitive and valuable tool for identifying recurrent chromosomal abnormalities.

Spectral karyotyping

chromosomes of a single cell

Pepsin treatment( at 37 degree C for 3-5 min)

labeled with a different combination of fluorescent dyes and allowed to hybridize, specific for each chromosome

Imaged immediately and Spectral karyotype done using SPK View software

SPK

Comparative genomic hybridization

2 genomes Test DNA

Normal DNA

Labeled with 2 different fluorescence(green and red) dyes

Allowed to hybridize

2 samples are equal focal deletion orProduce yellow fluorescence duplication

fluorescence skewed towards green or red.

• Uses - Has higher sensitivity - Can be performed using DNA extracted from

fixed as well as tumor sample - Technique makes it possible to perform a

genome wide scan for structural alteration even on those cases for which other cytological analysis is not feasible or successful .

CHROMOSOMAL ABNORMALITIES

NUMERICAL STRUCTURAL

Karyotype with abnormal no. of chromosomesInclude chromosome losses & gains

Alterations in structureInclude loss, rearrangementsor gain of chromosome segments

Numerical abnormalities

• Haploid- gametes 23 or N• Diploid- 46 or 2N• Euploid- exact multiple of N eg: 3N (triploid), 4N

(tetraploid) • Aneuploid- indicates noneuploid, loss or gain of

single chromosomes eg: monosomy, trisomy • Most common mechanism of aneuploidy-

nondisjunction of chromosomes

• Nondisjunction in Meiosis I, results in 2 gametes with parental chromosomes that fail to separate and 2 nullisomic gametes

• Nondisjunction in meiosis II,results in 1 gamete with two identical chromosomes, 1 nullisomic and 2 normal gametes.

• Monosome: fertilization of nullisomic with normal gamete

• Trisome: fertilization of gamete retaining both paternal and maternal or both copies of either maternal or paternal with normal gamete

• Mosaicism: Nondisjunction when occurs in mitosis, a condition where individual has two or more cell lines of different chromosomal constitution derived from same zygote.

Structural abnormalities

• Translocation- Reciprocal translocation- Robertsonian translocation

• Inversion - Paracentric inversion - Pericentric inversion

• Deletion - Terminal deletion - Inserstitial deletion

• Microdeletions : - Subtype of chromosome deletion that can be

observed only in banded chromosomes or in some cases using molecular genetic approaches.

• Duplications:- Intra chromosomal gain of chromatin lying in the same linear orientation (direct) reverse orientation (inverted ) with respect to

centromere.

• Insertion - Interchromosome insertion - Intrachromosome insertion - Direct insertion - Inverted insertion

• Isochromosomes – either two identical short arms or two identical long arms.

This occurs as a result of transfers split instead of longitudinal split during meiosis and mitosis.

• Ring chromosome- these are formed when a break occurs on each arm of chromosomes followed by fusion of the exposed ends to create a circular structure. The distal fragments are lost because they lack the centromere.

• Uniparental disomy:- A condition in which one parent has

contributed 2 copies of chromosome and other parent has contributed no copies.

- Ex: Prader-Willi syndrome Angelman syndrome

CYTOGENETIC ABNORMALITIES

1. Chromosomal disorders - Autosomes - Sex chromosomes 2. Cancer cytogenetics - Soft tissue tumors - Hematological disorders

DOWNS SYNDROME• John Langdon Down in1866• Trisomy of chromosome 21• 1 in 700 live births• Major cause of mental retardation • Maternal age has a strong influence – as the age

increases the risk of down syndrome increases

Trisomy 21 Karyotype and FISH

General Hypotonia with tendency to keep mouth open Protruding tongue

CraniofacialBrachycephaly with flat occiputMild microcephalyUpslanting palpebral fissuresLate closure of fontanellesAplasia of frontal sinusLow nasal bridgeInner epicanthal folds

Eyes Speckling of iris (Brushfield spots)Fine lens opacity,refractive errorNystagmus,strabismus,blocked tear duct

Ears Small,overfolding of upper helixSmall or absent ear lobesHearing loss

Skin loose folds in posterior neck (infancy)Cutis marmorata – extremities

Hands Short metacarpal and phalangesHypoplasia of mid phalanx of 5th fingerSingle palmar deep flexion crease-simian

crease

Feet Wide gap between 1st and 2nd toesrocker-bottom feet

Cardiac Endocardial cushion defects-40%

VSD,PDA,ASD,MVPAR by 20yrs of age

EDWARD SYNDROME• TRISOMY 18 SYNDROME• 1 per 6,000 newborn babies,<5% survive to term• 47,XY/XX+18• Second most common autosomal trisomy

Trisomy 18 syndromeKaryotype and FISH

General Prenatal growth deficiencyCraniofacial Characteristic facial features

Small ear,small mouth,Retrognathia

Hands and feet clenched hand,overlapping of fingersNail hypoplasia,short big toes

rocker bottom feet Thorax short sternum,small nipplesAbdominal wall unbilical hernia,small pelvis

Omphalocele-protrusion of bowel into umbilical cord

others VSD,cryptorchidism,hirsutism

PATAU SYNDROMETRISOMY 13 SYNDROME1 in every 5,000 births47,XY,+13Craniofacial Mental retardation

Microcephaly,microphthalmia,coloboma of irisCleft lip,palate/bothAbnormal helices,low set ears

Skin Capillary hemangioma,loose skinHands & feet Distal palmar triradii,flexion of fingers

PolydactylyCardiac VSD,PDA.Others Cryptorchidism,bicornuate uterus

Trisomy 13 syndromeKaryotype and FISH

TURNERS SYNDROME

• 45X SYNDROME - X0,• Complete or partial monosomy of X chromosome

characterized by hypogonadism in phenotypic females.

• Henry Turner – 1938• 1 in 2000 live born females.

Karyotyping in Turner

Classic facial features

Epicanthal foldsDown-slanting palpebral fissures Flat nasal bridgeReceding chinLowset earsExcessive nuchal skin folds.Webbed neck

KLINEFELTERS (XXY) SYNDROME

• Male hypogonadism • 2 or more X chromosomes and one or more Y

chromosomes.• Harry Klinefelter - 1942• Most common cause of hypogonadism and infertility• 1 in 500 males affected• Classic pattern – 47XXY karyotype in 82% of cases.• Other mosaic patterns – 46XY/47XXY, 47XXY/48XXXY,

48XXXY/49XXXXY.

Karyotype 47,XXY

Performance Normal to low IQDelayed speech,Poor memoryBehavioral problemsProblems with psychosocial adjustment

Growth Long limbs, Low upper to lower segment ratioTall and slim stature

Gonads Hypogonadism,HypogenitalismOthers Elbow dysplasia, FSH and Estradiol

Testosterone,Gynecomastia

XXXXX SYNDROMEPENTA X SYNDROME• First described by Dr.Nirmala kesaree and Wooly in

1963.• Found the abnormality in prisoners in America.

Karyotype of XXXXX

• Mental retardation• Prenatal growth retardation • Short stature• Microcephaly• Hypertelorism• Low set ears• Mongoloid slant• Abnormal behavior• Clinodactyly of fingers

CRI-DU-CHAT SYNDROME(5p-)

• Deletion of short arm of chromosome 5 (5p-)• Critical region : 5p15• 1 in 15,000 to 1 in 50,000

5p-

MicrocephalyRound faceHypertelorismEpicanthal foldsDownward slanting of palpebral fissuresStrabismus – often divergentLow set/poorly formed earsFacial asymmetry

Cat like cry- mewing of a cat, due to abnormal laryngeal development,become less pronounced with increasing age

Prader-willi syndrome• 1 in 15,000.• Mechanism:

Deletion of 15q at q11-q13(paternally derived)-75%Maternal UPD – 2 maternal,no paternal copies of 15q – 20%Chromosomal translocation involving proximal 15q – 5%

Craniofacial Almond shaped eyesUpslanting palpebral fissuresStrabismus Thin upper lip

Performance Mental retardation mild 63%,moderate 31%Excess appetiteobsession with eating

obesity Hands and feet Small

Narrow hands,straight ulnar borderGenitalia Small penis,cryptorchidism

Hypoplastic labia minora & clitorisHypogonadism

Angelman syndrome

• HAPPY PUPPET SYNDROME – abnormal puppet like gait,characteristic facies,paroxysms of laughter,due to brain stem defect – not apparently associated with happiness

• MechanismDeletion of 15q11 ( maternal origin )-75%Paternal uniparental disomy in 2%Imprinting mutation 2%

Happy disposition,an open mouth expression, widely spaced teeth, and a pronouncedmandible

• Confirmation of the diagnosis of CML• Confirmation of blast crisis of CML• Diagnosis of Acute leukemias• Diagnosis of lymphoproliferative

disorders• Diagnosis of non hodgkins lymphomas

Clinical applications of cytogenetics in hematological disorders

Chronic Myeloid Leukaemia-(Ph+)

• t(9:22)(q34;q11)/BCR-ABL abnormality- Philadelphia chromosome , identified in approximately 92 % of CML patients

Other abnormalities • Del(9q)• +8• i(17q)

Polycythemia vera (PV) • The most common anomalies are- +8,- –7, or a del(7q)- del(11q)- del(13q)- del(20q).

Acute Myeloid Leukaemia• Broadly classified as being favorable, intermediate or poor

prognostic types Eg –extensive numerical/structural karyotype abnormality aggressive myelodysplastic background t(15:17) t(8:21) favorable inv (16) or related t(16:16)

Acute Lymphoblastic Leukaemia• most common - t(9:22)• Tumor genetics →used for risk evaluation - hyperdiploidy(>52 chr) →favorabble - t(12:21) → favorable - t(1:19) → unfavorable - t(9:22)→ unfavorable - 11q23 translocation like t(4;11), t(11;19) → unfavorable - hypodiploidy(<40 chr) →unfavorable

Lymphoproliferative disorders • CLL- Only 50 % of CLL patients have detectabl chromosomal abnormalities- Trisomy 12 – more common – worse prognosis- Less often structural abnormalities seen – del(13q),

del(14q), del(17p)/- 17, del(6q)• Multiple myeloma structural abnormalities of chromosome- t(14;16),

t(4;14)

Non hodgkin’s lymphoma(NHL)

• t(14;18) in follicular lymphoma • t(8;14), t(2;8) and t(8;22) in Burkitt lymphoma• t(11;14) in mantle cell lymphoma• t(3;22)or t(3;14) in diffuse large B-cell

lymphomas (DLBCL)• t(2;5) or t(1;2) in Anaplastic large cell

lymphoma (ALCL)

NEED OF CYTOGENETICS IN SOFT TISSUE TUMORS

• Understanding of soft tissue tumor biology • A substantial set of soft tissue tumors contain

specific karyotypic abnormalities and thus helps in diagnosis

• Provide insight into pathogenesis, classification, prognostic factors.

A Karyotype from a lipoma shows the most common rearrangement t(3;12)(q27;q15)

Karyotype of a Ewing’s tumor showing translocation of chromosome 11 and 22, chromosome 3 on right side is shorter than its partner because of a deletion.

Karyotype of a benign schwannoma with monosomy of chromosome 22

Complex Karyotype of a malignant peripheral nerve sheeth tumor showing aneuploid with numerous chromosomal gain, losses and rearrangement.

Karyotype of dermatofibrosarcoma protuberans showing supernumerary ring chromosome.

Conclusion

• Bridge JA, Sandberg AA. Cytogenetics. In: Andersons pathology. 10th ed. Mosby publications; 1996. p.223-57.

• Chen H. Atlas of genetic diagnosis and counselling. New jersy: Humana press publishers; 2006.

• Kuman V, Abbas AK, Fausto N. Genetic disorders. In: Robbins and cotran pathologic basis of disease. 8th ed. Philadelphia: Elsevier publication; 2004. p.145-92.

• Rimoin DL, Connor JM, Pyeritz RE, Korf BR. Chromosomal disorders. In: Principles and practice of medical genetics. 4th ed. London(UK): Churchill livingstone; 2002. vol 1. p.1129-1202

• Fletcher AJ. Cytogenetic analysis of soft tissue tumors. In: enzinger’s and weiss’s soft tissue tumors. 5th ed. Kathleen neely ,jhon canelon(USA, UK); 2008. p. 125-141

• Kaushansky k, litchman AM, Beutler E, Kipps TJ, Seligsohn U, Prchal TJ. Cytogenetics and molecular abnormalities. In: wiiliams hematology 8th ed. United states; 2010 .