cytogenetic abnormalities dr dhanya a n sri siddhartha medical college, tumkur

1. Cytogenetic abnormalities
Presenter -Dr Dhanya A N
Moderator–Dr chaithra

2. 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

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

4. 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.

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

6. Types of chromosomes

7. CELL CYCLE
• Interphase – G1, S, G2
• M phase (Mitosis)
• G0 – NO cell division

11. 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.

12. 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.

13. APPROACH TO THE DIAGNOSIS OF
CYTOGENETIC DISORDERS
1. Karyotyping
2. Insitu hybridization
3. Fluorescence insitu hybridization
4. Spectral karyotyping
5. Comparative genomic hybridization

14. 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.

15. 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)

16. • 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

17. 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

18. 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) .

19. 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

21. 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 .

22. 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

23. 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.

24. 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.

25. 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)

27. 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.

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

30. 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

31. ISCN of chromosome 11

32. 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.

33. • 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.

34. 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

36. 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.

37. 2] Locus specific
identifier(LSI) probe
Target distinct
chromosomal region of
interest and utilize single
copy rather than
repetitive DNA.

38. 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

39. 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

40. 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.

41. 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

42. SPK

43. 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
or
Produce yellow fluorescence duplication
fluorescence skewed
towards green or red.

45. • 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 .

47. CHROMOSOMALABNORMALITIES
NUMERICAL STRUCTURAL
Karyotype with abnormal no. of
chromosomes
Include chromosome losses & gains
Alterations in structure
Include loss, rearrangements
or gain of chromosome segments

48. 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

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

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

51. • 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.

52. Structural abnormalities
• Translocation
- Reciprocal translocation
- Robertsonian translocation

53. • Inversion
- Paracentric inversion
- Pericentric inversion

54. • Deletion
- Terminal deletion
- Inserstitial deletion

55. • Microdeletions :
- Subtype of chromosome deletion that can be
observed only in banded chromosomes or in some
cases using molecular genetic approaches.

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

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

58. • 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.

59. • 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.

60. • 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

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

62. 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

63. Trisomy 21
Karyotype and FISH

64. General Hypotonia with tendency to keep mouth open
Protruding tongue
Craniofacial Brachycephaly with flat occiput
Mild microcephaly
Upslanting palpebral fissures
Late closure of fontanelles
Aplasia of frontal sinus
Low nasal bridge
Inner epicanthal folds

65. Eyes Speckling of iris (Brushfield spots)
Fine lens opacity,refractive error
Nystagmus,strabismus,blocked tear duct
Ears Small,overfolding of upper helix
Small or absent ear lobes
Hearing loss
Skin loose folds in posterior neck (infancy)
Cutis marmorata – extremities

66. Hands Short metacarpal and phalanges
Hypoplasia of mid phalanx of 5th finger
Single palmar deep flexion crease-simian
crease

67. Feet Wide gap between 1st and 2nd toes
rocker-bottom feet
Cardiac Endocardial cushion defects-40%
VSD,PDA,ASD,MVP
AR by 20yrs of age

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

69. Trisomy 18 syndrome
Karyotype and FISH

70. General Prenatal growth deficiency
Craniofacial Characteristic facial features
Small ear,small mouth,
Retrognathia

71. Hands and feet clenched hand,overlapping of fingers
Nail hypoplasia,short big toes
rocker bottom feet
Thorax short sternum,small nipples
Abdominal wall unbilical hernia,small pelvis
Omphalocele-protrusion of bowel
into umbilical cord
others VSD,cryptorchidism,hirsutism

72. PATAU SYNDROME
TRISOMY 13 SYNDROME
1 in every 5,000 births
47,XY,+13
Craniofacial Mental retardation
Microcephaly,microphthalmia,coloboma of iris
Cleft lip,palate/both
Abnormal helices,low set ears
Skin Capillary hemangioma,loose skin
Hands & feet Distal palmar triradii,flexion of fingers
Polydactyly
Cardiac VSD,PDA.
Others Cryptorchidism,bicornuate uterus

73. Trisomy 13 syndrome
Karyotype and FISH

75. 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.

76. Karyotyping in Turner

77. Classic facial features
Epicanthal folds
Down-slanting palpebral fissures
Flat nasal bridge
Receding chin
Lowset ears
Excessive nuchal skin folds.
Webbed neck

78. 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.

79. Karyotype 47,XXY

80. Performance Normal to low IQ
Delayed speech,
Poor memory
Behavioral problems
Problems with psychosocial
adjustment
Growth Long limbs,
Low upper to lower segment ratio
Tall and slim stature
Gonads Hypogonadism,Hypogenitalism
Others Elbow dysplasia, FSH and Estradiol
Testosterone,Gynecomastia

81. XXXXX SYNDROME
PENTA X SYNDROME
• First described by Dr.Nirmala kesaree and Wooly in
1963.
• Found the abnormality in prisoners in America.

82. Karyotype of XXXXX

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

84. 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

85. 5p-

86. Microcephaly
Round face
Hypertelorism
Epicanthal folds
Downward slanting of palpebral
fissures
Strabismus – often divergent
Low set/poorly formed ears
Facial asymmetry
Cat like cry- mewing of a cat, due to
abnormal laryngeal development,become
less pronounced with increasing age

87. 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%

88. Craniofacial Almond shaped eyes
Upslanting palpebral fissures
Strabismus
Thin upper lip
Performance Mental retardation mild 63%,moderate 31%
Excess appetite
obsession with eating
obesity
Hands and feet Small
Narrow hands,straight ulnar border
Genitalia Small penis,cryptorchidism
Hypoplastic labia minora & clitoris
Hypogonadism

90. Angelman syndrome
• HAPPY PUPPET SYNDROME – abnormal puppet like
gait,characteristic facies,paroxysms of laughter,due
to brain stem defect – not apparently associated with
happiness
• Mechanism
Deletion of 15q11 ( maternal origin )-75%
Paternal uniparental disomy in 2%
Imprinting mutation 2%

91. Happy disposition,
an open mouth expression, widely
spaced teeth, and a pronounced
mandible

92. • 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

93. 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)

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

97. 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)

99. 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

100. 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)

101. 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)

102. 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.

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

105. 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.

106. Karyotype of a benign schwannoma with monosomy of chromosome 22

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

108. Karyotype of dermatofibrosarcoma protuberans showing
supernumerary ring chromosome.

109. Conclusion

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