Cytogenetics

2005
 Cytogenetics
 The study of chromosome
and the related disease
states caused by
abnormal chromosome
number and\or structure.
 Chromosomes : complex
structures located in the
cell nucleus, composed of
DNA, histone and non-
histone proteins, RNA,
and polysacchairdes.
 History of human cytogenetics
 “Dark Ages’’ ( prior to
1952 )
no. of chromosomes = 48.
 The “Hypotonic Era”
started in 1952
no. of chromosomes = 46.
 The “Trisomy Period”.
 The “Banding Era”.
 The “Molecular Era”.
Cell Division – Meiosis I& II
Karyotype preparation
 Chromosomes Banding
Effect
Under UV light, distinct
fluorescent banded pattern for
each chromosome.
Distinct banded pattern for each
chromosome; same as Q-
banding pattern except single
additional band near centromere
of chromosomes 1 and 16
Reverse banding pattern of that
observed with Q- or G-banding
Largest bands usually on
chromosomes 1, 9, 16, and Y;
chromosomes 7, 10, and 15 have
medium-sized bands; size of C-
bands highly variable from
person to person
Area Stained Stain Type
Chromosome arms; mostly Quinacrine Q-banding
repetitive AT-rich DNA
Chromosome arms; mostly Giemsa G-banding
repetitive AT-rich DNA
Chromosome arms; mostly unique Variety of techniques R-banding
GC-rich DNA
Centromere region of each Variety of techniques C-banding
chromosome and distal portion of Y
chromosome; highly repetitive,
mostly AT-rich DNA
Non-Banded Karyotype
G-Banding/chromosome
morphology
Ideograms
Chromosome Morphology
Normal Karyotype
Low/High Resolutions Karyotype

18
7
Q-Banding
C-Banding
R-Banding
Changes in number, or sets, of chromosomes
A) Polypoidy – change in complete sets of chromosomes
(3n, 4n, etc)
plants > animals.
B) Aneuploidy – change in the no. of chromosomes
 nullisomy 2n-2
 monosomy 2n-1
 trisomy 2n+1
 tetrasomy 2n+2
Gene dosage effect
1- Sex-chromosomal aneuploids .
2- Autosomal aneuploids .
 Changes in structure of individual
chromosome
A) Chromosome rearrangements:
Effects
 Deletion. pseudodominance
dicentric chr.
 Duplication. gene dosage
 Inversion – paracentric positional
pericentric
 Translocation. positional
new linkage rearrangement

B) Fragile-X Syndrome.
C) Cancer/mutations.
Fragile-X-Syndrome
 Advantages and Disadvantages of conventional technique

 Advantages
1- Enable the entire genome to be viewed at one time.
2- Suitable when a specific anomaly is suspected ( e.g.
Philadelphia in CML ) and as a general diagnostic tool
to detect additional chr. Abnormalities commonly
seen in disease progression of CML.
 Disadvantages
1- Detect major structural abnormalities
( one band = 6mb of DNA ~ 150 genes ).
2- Labor intensive and highly dependent upon operator
experience and skills.
 Fluorescence in situ hybridization
(FISH)
 Increased the sensitivity ,
specificity ,and resolution of
chromosome analysis.
 Fluorescently labeled DNA
probe ~40 kb.to detect or
confirm gene or chromosome
abnormalities that are
beyond the resolution of
routine cytogenetics.
 Metaphase FISH
 Interphase FISH
Metaphase- FISH
Interphase-FISH
 Fluorescence in situ hybridization (FISH)
I. Microdeletion Syndromes
 Cri-du-chat (5p-).
 Miller-Dieker syndrome (7q11.23).
 Smith-Magenis syndrome (17p13.3).
 Steroid Sulfatase Deficiency (Xp22.3).
 DiGeorge/Velo-cardio-facial/CATCH-22/ Shprintzen
Syndrome (22q11.2).
 Kallman Syndrome (Xp22.3).
 Williams Syndrome (7q11.23).
 Wolf-Hirsch horn (4p-).
 Prader-Willi/Angelman Syndrome (15q11.2-13).
 X-Linked Icthyosis (xp22.3).
 Retinoblastoma (13q14).
Di-George Syndrome
 II. Trisomy Detection and Sex
Determination

Probes for
chromosomes
13,18,21,X,Y
.and SRY
III. Oncology

Single Gene Probes( deletion or amplification)

.P58 CLK-1 Locus (1p36)
.D7S486 (7q31)
.Retinoblastoma (13q14)
.P53 (17p13.1)
.Her-2/ neu (17q11.2-q12)
.Oncology-cont

Enumeration probes for all chromosomes

Dual Color Translocation Probes.
bcr/abl translocation t(9;22)(q34;q11.2).
M-bcr/abl translocation t(9;22)(q34;q11.2).
IGH/CCND1 translocation t(11;14)(q13;q32).
PML/RARA translocation t(15;17)(q22;q21.1).
TEL/AML1 translocation t(12;21)(p13;q22).
Fluorescence hybridization in situ(FISH)
. cont
:Prenatal/Neonatal screening

Aneuploid detection by FISH

for chromosomes
.13,18,21,X,Y

:Telomere Alteration Testing

Identify alterations in 7-10%

of cases with MR and
multiple congenital anomalies
.with mental retardation
 .FISH-cont
:Advantages
 The resolution is better (L ~ 2mb).
 Can be applied to both dividing and
non-dividing cells.
 The tech. is straightforward.
 Hybridization with multiple probes enable
detection of translocation products.
 Can identify a range of mutations.
 Monitor recurrent or residual disease
in BMT pt.
 .FISH-cont

:Disadvantages
 Cannot detect small mutations.
 Miss Uniparental disomy.
 Miss Inversions.
 Probes are not yet commercially

.available for all chromosomal regions

Spectral Karyotyping (SKY) and Multiple
Fluorescence In Situ Hybridization(M-FISH)

Simultaneous visualization of all

human (or mouse)
Chromosomes in different colors.
A combination of five
fluorochromes to paint all 22
autosomes,and the X and the
Y. then analyzed based on
their particular emission
.spectra
Spectral Karyotype of human
chromosomes
Ewing Sarcoma
 SKY-cont.
:Advantages
 Mapping of chromosomal breakpoints.
 Detection of subtle translocations.
 Identification of marker chromosomes,homogeneously
staining regions,and double minute chromosomes.
 Characterization of complex rearrangements.
:Disadvantages
 Very expensive equipments.
 The technique is labor intensive.
 Dose not detect structural rearrangements within
a single chromosome.
 Low resolution (up to 15 mb ).
 Specific, not a screening method.
Genomic Comparative Hybridization(CGH)

Fluorescent molecular tech. that identifies DNA

gains,losses,and amplifications (mapping to)metaphase
.chromosomes
Based on quantitative two-color FISH
.)FITC for tumor DNA and TRITC for the normal DNA(
:Advantages
 Require only genomic tumor DNA.
 Can be applied to fresh or frozen tissues,cell lines,
and archival formalin-fixed paraffin-embedded
samples.
Comparative Genomic Hybridization(CGH)
Comparative Genomic Hybridization(CGH)
Genomic Comparative Hybridization(CGH)
Comparative Genomic Hybridization(CGH)
Genomic Comparative Hybridization(CGH)

Disadvantages:
 Cannot detect balanced abnormalities.
 Chromosomal copy changes < 10 mb. are not
resolved.
 Copy no. changes < ½ of the analyzed cells are
not detected.
 Diagnostic Potential For Karyotype, FISH, and Chromosomal
Micro- array Analysis (CMA) For Selected Disorders
CMA Telomere FISH Disease Karyotype Locus Condition
specific FISH studied

~100% Detected by Not detected ~100% various Aneuploidy

karyotype
Karyotype Detected by Not detected ~100% various Large deletions, large
better for karyotype dupllications, translocation of
present large segments
~100% for ~100% Not detected Not various Cryptic
unbalanced detected Rearrangements of telomeres
~99% >95% ~99% Few 1p36.3 1p36 deletion

~99% >95% ~99% Most 4p16.3 Wolf-Hirschhorn

~99% >95% ~99% Most 5p15.2 Cri-du-chat

~99% Not detected ~99% Almost 7q11.2 Williams-Beuren

none
~70% Not detected ~70% Unreliable 15q11-q13 Prader-Willi

~70% Not detected ~70% Unreliable 15q11-q13 Angelman

>90% Some detected >90% Few 17p13.3 Miller-Dieker lissencephaly

>95% Not detected >95% Some 17p11.2 Smith-Magenis

>95% Not detected >95% Rarely 22q11.2 Velocardiofacial/DiGeorage 1