Cytogenetics
Mgr. Jan Smetana
Department of experimental biology, Faculty Of Science, Masaryk University Brno
Laboratory of Molecular Cytogenetics, Babak Research University, Brno
What´s the story today?
1. Historical overview of cytogenetics
2. Current techniques and methods used in cytogenetics
3. Utilization of cytogenetics in clinical praxis
What is cytogenetics
• The branch of biology that deals with heredity and the
cellular components, particularly chromosomes, associated
with heredity.
or more precisely we could say
• Cytogenetics is branch of the genetics, focusing on the study
of chromosomes (the number, morphology, numerical and
structural abnormalities, segregation in normal and
pathological conditions) and the correlation of these findings
with the phenotype
How and when has it begun ?
• J. and Z. Jansen (1590)
Father and son from Midlenndburg (Netherlands), experiments with lenses,
first microscope = tube with lenses on both sides
• G. Galiley (1564 – 1642)
Improvements in Jansen´s construction
• A. van Leevenhook (1632 – 1723)
„father of microbiology“
He constructed first trully microscope, used for biology
1676 – fisrt observation of bacteria under microscope „Animalcules“
19th century
• Two major events in the mid-1800’s led directly to the
development of modern genetics.
• 1859: Charles Darwin publishes The Origin of
Species, which describes the theory of evolution by
natural selection. This theory requires heredity to
work.
• 1866: Johann Gregor Mendel publishes his
Experiments in Plant Hybridization, which lays out
the basic theory of genetics.
Augustinian monks of St. Thomas
monastery in Brno
Gregor Mendel
defined basic principles of the heredity
principle of segregation
principle of combination
Law of Segregation
„during gamete formation allele pairs separate
or segregate, into different gametes“
Law of Independent Assortment
suggested that each
allele pair segregates
independently of other
gene pairs during
gamete formation
20th century I.
• During his live, Mendel´s work was widely ignored
• 1900: rediscovery of Mendel’s work by Robert
Correns, Hugo de Vries, and Erich von Tschermak .
• 1904: Gregory Bateson discovers linkage between
genes. Also coins the word “genetics”.
• 1910: Thomas Hunt Morgan proves that genes are
located on the chromosomes (using Drosophila).
Thomas Hunt Morgan
(September 25, 1866 – December 4, 1945)
Sex linked inheritance of the white eyed mutation.
Source: Wikipedia, 2010
20th century II.
• 1926: Hermann J. Muller shows
that X-rays induce mutations.
• 1944: Oswald Avery, Colin
MacLeod and Maclyn McCarty
show that DNA can transform
bacteria, demonstrating that DNA
is the hereditary material.
• 1953: James Watson and Francis
Crick determine the structure of the
DNA molecule, which leads directly
to knowledge of how it replicates
Joe Hin Tjio (1919 - 2001)
Human somatic cells nuclei contain 23 pairs of
chromosomes, thus overall 46 chromosome
Albert Levan (1905 -1998)
Tjio, T.H., Levan, A.:
The chromosome number of man.
Hereditas 42:1, 1956
Basic conditions for development
of human cytogenetics
• improved techniques of cells cultivation in vitro
• use of hypotonic solution (0.075 M KCl)
• establishing squash techniques
• use of colchicine – arrest of mitotic division
• 1% orcein staining
Development of human cytogenetics
• „Dark Ages" - the development and improvement of tissue culture techniques
• "Hypotonic Period“
- hypotonization of cell samples (1951 - 0,075 m KCl)
- using phytohaemagglutinin (PHA) - stimulation of peripheral blood lymphocytes - 1960
• "Trisomy Period - trisomy of chromosome 21-1959
• The first deletion syndrome - "Cri du chat" - 1963
• "Banding Area - chromosome banding techniques 1968 – 1970
• "Molecular Area" –
- in situ hybridization technique – 1970
- FISH – 1986
- Comparative genomic hybridization (CGH) - 1992
- Spectral karyotyping (M-FISH, SKY) - 1996
- M - banding - 2001
- Array - CGH - molecular karyotyping
Nomenclature of human chromosomes
1960: Denver Conference - sort of human chromosomes into groups
according to size and shape
1963: London Conference - chromosomes are sorted into 7 groups,
sign A – G
1966: Chicago Conference - the description of chromosome changes
1971: Paris Conference - the identification and labeling of
chromosomes using banding techniques
An International System for Human Cytogenetic Nomenclature
(ISCN 1978)
Examples of cytogenetic findings in
patients enrolled under the rules of ISCN
• 46,XX or 46,XY; healty female or male
• 47,XX,+21 Down s.
• 47,XY,+18 Edwards s.
• 47,XY,+13 Patau s.
• 46,XY,del(13q)
• 45,XY,-13
• 45,X,-Y
• 46,XY,t(2;5)(q21;q31)
• 44,X,-X,der(1),del(4p),-11,der(12),-16,-22,+2mar
• 44,XX,+3,del(6q),der(8),del(10q),-13,-16
• 46,XY,-3,der(12)t(3;12)
• 47,XY,der(1),der(4),+9,-13,+20
• 46,XY,del(5q)
• 49,XY,+7,+10,+17
• 44,XY,-1,-10,der(11),-13,del(14q),-22,+2mar
• 44,XY,der(1),der(5),der(6),der(9),del(13q),der(15),-22,-22
• 62,XY,+2,+3,+5,+5,+6,+7,+7,+9,+10,+11,+14,+16,+17,+20,+20,+3mar
Craig Venter
Head of Celera Genomics
Francis Collins
Head of the Human Genome Project
Human Genome Project & cytogenetics
Human Genome Project & cytogenetics
•Chromosomome 22 - first “decoded”
chromosome (1999) contains 450 genes
•Chromosomes 19 a 22 - the biggest density of
genes per Mb
•Chromosomes 13 a Y the lowest number of
genes / Mb
Nowadays and future
• Next (second) and third (single molecule) generation sequencing
• SOLiD (ABI), SOLEXA (Illumina), 454 pyrosequencing (Life Science)
• Key challenge = sequencing whole human genome during 24 hours for less than
$1000
Techniques and methods used for
cytogenetic analyses in OLG Brno
Chromosome morphology
Chromosome classification according to
position of the centromere
• Metacentric chromosomes
centromere entirely or almost entirely in the
middle, short and long arms are
(almost) the same length
• Submetacentric chromosomes
centromere of chromosome off-center, p
q arms are clearly distinguished by length
Chromosome classification according to
position of the centromere
Acrocentric chromosomes
• centromere is located very close to one end
from the short arms are constricted so
called satellites
• constricted position = secondary constriction
• secondary constriction contains copies of
genes coding for rRNA nucleolar organizer
=NOR
ISCN classification according to size and
shape of chromosomes
Group 1-3 (A) Large chromosomes
with approximately
median centromeres;
1,2, and 3 can usually
be identified
morphologically.
Group 4-5 (B) Large submetacentric
chromosomes.
Group X, 6-12(c) Medium sized
submetacentric
chromosomes.
Group13-15(D) Large acrocentric
chromosomes.
Group 16-18(E) No.16 is metacentric;
No.17-18 are small
sub-meta-centric
chromosomes.
Group 19-20(F) Small metacentric
chromosomes.
Group 21-22 + Y(G) Short acrocentric
chromosomes.
(The Y chromosome
belongs to this group,
but has no satellites;
it is of variable size
and can usually be
identified
morphongenically.
Bands on each arm are numbered in ascending
order from centromere to telomere
Band numbering of chromosomes
Number of band allows its unique identification
1.rozpruhování
2.rozpruhování
with progressive chromosome condensation
reduces the number of bands
Band numbering of chromosomes
Source of material for cytogenetic
investigation
• peripheral blood
• samples from different tissues
• amniotic fluid cells, chorionic villi, placenta
umbilical cord blood
• Bone marrow
• samples of solid tumors
Source of material for
cytogenetic investigation
Solid tumorPeripheral blood Bone marrow
Sample preparation
According to type of investigation we could
1.Cultivate cells – obtaining mitosis
2.Sort just specific types of cells – MACS,
FACS
3.DNA or RNA extraction
Sample preparation
Cultivation of amniotic fluid cells
bone marrow
gradient
centrifugation
magnetic
separation
gradient
centrifugation
FACS
Aria
erythrolysis
FACS
Aria
Sample preparation
Sorting of sample cells
MACS – magnetic activated cells sorting
FACS – fluorescent activated cell sorting
Isolation of nucleic acids
• Phenol – chloroform extraction
• Isolation using commercial kits
Methods for identification of chromosomal
changes used in cytogenetics
A)Conventional staining (acetoorcein, Giemsa)
B)Banding technology (processing chromosomes +
Giemsa or fluorochromes ....)
C)molecular cytogenetics using DNA probes
G-banding
• Caspersson et al. 1968
- using fluorescent dye (chinakrine yperit) make
specific bands on chromosomes = banding
Banding techniques
• Bands = stripes on the chromosomes, which are clearly
distinguishable from adjacent segments
• Methods of differentiating along the length:
Q-bands
G-bands
R-bands
• Selective methods:
C-bands
T- bands
NOR staining
Human karyotype: G-banding
C-bands: detection of centromeres
Staining and structure of the human
genome
• bands on chromosomes reflects the structure of the genome and its
functional organization
• Each band contains 5 to 10 Mbps
• Giemsa positive bands (G +, rich in AT pairs, the late replicating
chromosome regions poor in genes)
• Giemsa negative bands (G-rich in GC pairs, the early replicating
chromosome regions rich in genes)
Molecular cytogenetics
• Presents the connections between classical cytogenetics
and molecular biology
• utilizes the latest knowledge of molecular biology,
microscopy and computer image analysis to study the
structure and properties of chromosomal changes
• allows the analysis of numerical and structural chromosomal
imbalances unidentified classical cytogenetic techniques
• does not require the presence of mitosis
Principle of in situ hybridization
FISH
(fluorescent hybridizace in situ)
Basic equipment for FISH analysis
• Fluorescent microscope
with appropriate
objectives and fluorescent
filters
• High sensitive CCD
camera
• Suitable PC + software for
picture analysis
Analyses using FISH
• Based on detection of the fluorescent signals
through microscope equipped with specific
fluorescent filters
• We determine:
1. Presence of the signals
2. Number of signals
3. Position of signals
Spectral karyotyping (SKY)
• Each chromosome is labeled with a unique combination
of the five fluorescent dyes.
• This results in a unique fluorescence SPECTRUM of
each chromosome.
Comparative Genomic Hybridization
(CGH)
• Screening and mapping unbalanced genetic
alterations (deletions, duplications) across
whole genome in single experiment
• Huge amount of data, detection changes
smaller than 5kb
Original CGH
Kallioniemi et al. 1992
detection range: 5 - 10 Mb
Kirchhoff et al. 1997
detection range: from 3 Mb
High resolution CGH
Array CGH
• Solinas-Toldo et al., 1997
New approach of scanning – not using
chromosomes, but BAC clones =
detection range from ~ 0,5 Mb
• Nowadays – BAC clones replaced by short (60 mers)
oligonucleotide probes utilized on array (spots)
• According to density of probes, detection range starts at 5kb
(1M Agilent array)
DNA of pacient
control DNA
loss
Genomic
clones
gain
CGH-Arrays
Pinkel et al., Nat Genet (1998), 20(2):207-11
Agilent Workbench Standard 5.1
All previously showed techniques allow:
- specify diagnosis
- determining and monitoring of
treatment strategies
- estimates of the likely development of
disease
In very fast way (24 hours average, 48 hours
for array-CGH)