Transcript Cytogenetic method

Methods of chromosomal study,
postnatal and prenatal cytogenetic
diagnosis
RNDr Z.Polívková
Lecture No 437 - course: Heredity
History of chromosomal study:



1903 - Sutton, Boveri – chromosomes are related
to heredity
1956 - Tjio-Levan – chromosome number in man = 46
1959 - Lejeune - 1st chromosomal abnormality =
trisomy 21 in Down syndrome
Methods of chromosomal study –
cytogenetic methods
Methods of clinical cytogenetics:
Postnatal methods: Cultivation of peripheral blood
- blood of affected persons and their relatives

Full blood (with anticoagulant heparine) is added to
cultivation medium with serum, PHA
(phytohemagglutinin PHA = mitogen – stimulates
T-lymphocytes to division)

Cultivation 48 or 72 hours = short term cultivation

cell division is stopped (in metaphase) by colchicine,
centrifugation of cell suspension

hypotonization of sediment - centrifugation


repeated fixation/centrifugation (3x)
preparation of slides and staining
Fixation-centrifugation
3x=washing
Slide preparation
centrifugation
Fixation
(methanole/acetic acid)
centrifugation
Medium
Serum
PHA
blood
Antibiotics
Hypotonization
0,075MKCl
Cultivation
72h
colchicine 1,5h
Cytogenetic method – peripheral lymphocytes
Indications to postnatal chromosomal
examination !!! (from peripheral blood – lymphocytes):
1. Specific phenotype (Down syndrome……)
2. Psychomotoric retardation (PMR), growth
retardation, dysmorphic features, congenital
malformations, small stature in girls, oedema in
newborns (TS)
3. Dysfertility (repeated spontaneous abortions, sterility
– chromosomal examination of both partners)
4. Amenorrhea, delayed puberty, genital malformations
5. Relatives of patient with chromosomal aberration
Prenatal methods: Detection of fetal karyotype:
1.
Cultivation of amniotic cells (AMC)
Collection of amniotic fluid at 16th week of pregnancy (approx.20ml)
=standard amniocenthesis (early amniocenthesis –

collection before 15th week)
long term cultivation of amniotic cells (it takes from 8 to14 days)
- cells are growing attached to the flask bottom and form colonies,
if there is several colonies in flask - division is stopped by colchicine
and cells removed from surface by trypsin

cytogenetic procedure:
hypotonization, repeated fixation etc.
AMC: reliable results
Cultivation of amniotic cells (AMC)
long term cultivation - colonies of dividing cells are attached
to the flask bottom, detaching of cells from surface by trypsin
Cultivation flask with
colonies of cells
Cells growing attached to the
bottom of cultivation flask-fibroblasts
Chorionic villi examination (CVS)



Collection of CVS at 10th week of pregnancy
Direct method or long term cultivation
Snipping cleaned tissue, detaching of cells by trypsin,
setting cells for cultivation
Chorionic villi = extraembryonal tissue – risk of karyotype
discrepancy !!!
It is better to combine both direct and cultivation methods,
or verify pathologic result (if only direct method is used) by different
method (fetal blood) or by detection of abnormality by ultrasonography
Gardner, Sutherland: Chromosome abnormalities and genetic counseling, 1996
Gardner, Sutherland: Chromosome abnormalities and genetic counseling, 1996
3. Cultivation of fetal blood



Similar procedure as cultivation of peripheral blood
Blood sample is collected from the loop of umbilical cord
Short term cultivation (48 h) and cytogenetic procedure
Used to verify some vague result of previous examination,
in case of abnormal finding of CVS examination (esp. if only
direct method is used), or in case of late detection of
abnormality on ultrasound
Indication of prenatal cytogenetic examination!!!
From cells of amniotic fluid, chorionic villi, fetal blood
1. Increased maternal age (≥ 35 years)
2. Patological values of biochemical markers
Screening in the 2nd trimester: „triple test“ = 3 biochemical markers:
AFP = α-fetoprotein
βhCG = choriogonadotropine
uE3 = estriol
Screening in the 1st trimester: markers – PAPP-A (pregnancy associated
plasma protein A), free -hCG
Combined screening in the 1st trimester - PAPP-A, free -hCG +
ultrasonography (nuchal translucency, nasal bone)
performed at 10th-13th week
Integrated test: biochemical markers of the 1st trimester + ultrasonography
markers + biochem.markers of the 2nd trimester (at 15th-17 th week)
highest effectivity, lowest degree of false positivity
1. Abnormality detected by ultrasonography (including small
morphological markers – e.g. nuchal translucency, nasal bone – combined
with 1st trimester biochemical markers)
2. One parent is a carrier of balanced chromosomal
aberration
3. Psychological indication (previous pregnancy with trisomy)
Staining:

Classic method =
homogenous staining by Giemsa
– for mutagenic study

Banding methods: differentiation of individual
chromosomes and their parts
- for detection of numerical and structural
chromosome abnormalities in clinical cytogenetics
Classic method for detection of
induced aberrations
Dicentric chromosome + difragment

G-bands: trypsin or saline solutions – different
denaturation of chromosomal parts = different staining
by Giemsa (dark and pale bands)

R-bands = reverse banding (reverse to G bands)
saline solutions at high temperature

C-banding : strong denaturation of euchromatic parts
(HCl, BA(OH) + saline solutions and high temperature)
only resistant heterochromatine is darkly stained,
strongly denaturated euchromatic parts are pale
- for study of heterochromatic variants

NOR (silver) staining – staining of active NORs
(producing rRNA) with silver nitrate
Banding method – G-bands
trypsine solution
in buffer pH 6,8
buffer
Giemsa solution
Karyotype of woman 46,XX – G banding
Karyptype of man - 46,XY – G bands
R- banding
C- banding
Y chrom.
FISH methods (fluorescent in situ
hybridization)
hybridization of probes (stained with fluorochromes)
exactly on cytogenetic slides (on the whole
chromosomes or interphase nuclei)

suitable for detection of small or complex structural
rerrangements, microdeletions

suitable for detection of chromosomal changes
connected with cancer also in interphase cells (fused
genes – e.g. fused gene bcr/abl, amplifications)

suitable for detection of aneuploidies in interphase
nuclei (without cultivation)
Probes:

- satellite= centromeric – for chromosome
counting, detection of „marker“ chromosomes =
chromosomes of unknown origin and detection of
aneuploidies in non-divided cells

locus specific – detection of microdeletions,
oncogenes (amplified or fused)

painting – for structural rearrangements, painting of
the whole chromosome
Probes
Signal localization
Examples of using
Detection of aneuploidy,
marker chromosomes…..
α-satellite
(centromeric)
G1
locus-specific
(gene)
G2
detection of
microdeletions,
oncogenes…
Principle of FISH methods
Chromosome or gene is marked with probe =
Probe = short sequence of DNA, labelled with fluorescent dye, complementary to
specific region on chromosome (gene, group of genes, chromosomal part or
whole chromosome)

DNA
Probe
denaturation of probe and
DNA examined
hybridization of DNA
with labelled probe
Detection of chromosome number by -satellite probe
Monosomy of chr.No 1 – cultivated cells of breast carcinoma
Detection of chromosome number by -satellite probe
Tetrasomy of chrom.No 11 - cultivated cells of breast carcinoma
Detection of oncogene amplification by locus specific probe
Amplification of Her2-Neu oncogene (red signals) - cultivated cells from metastase of breast carcinoma
Detection of oncogene amplification by locus specific probe
Amplification of Her2-Neu oncogene (red signals) - cultivated cells from metastase of breast carcinoma
Normal
cell
homozygous
loss of 9q21
(yellow signal
missing)
trisomy 7
green signals
Centromeric and locus specific probes for detection of numerical changes
and homozygous loss of 9q locus in urine bladder Ca (Wysis)
locus specific probes for detection of homozygous loss of 9q locus in
urine bladder Ca (Wysis) - (yellow signal is missing in some cells)
Detection of fused genes
bcr/abl by locus specific
probes (Wysis)
Detection of microdeletion by locus specific probe (Wysis)
Painting probes
(Wysis)
Detection of structural rearrangement by painting probes
M FISH
M - band
Non-cytogenetic methods
Microarray analysis = comparative genomic hybridization using
microarray as substrate (instead metaphase spread)
Microarray = thousands od spots of reference DNA sequences on a slide
Patients and control DNA (stained by different fluorescent dyes) are
applied to the slide to hybridize.
Excess chromatin is marked by red color (i.e.duplication of segment),
deficient hybridization is marked by green colour (i.e.deletion), even
amount of hybridization is yellow (overlapping od equal amounts of red
and green colour (i.e.normal segment)
The pattern of colour spots → analysed by computer
Method can detecet only unbalanced rearrangements
Cytogenetic methods in genotoxicology
- detection of acquired chromosomal aberrations
- can detect mutagenic exposure
e.g. dicentric chromosomes –used for biological dosimetry of radiation
exposure
Cytogenetic methods:
Metaphase analysis: - „classic“ method
- SCE (sister chromatid exchanges)
- FISH
Interphase analysis: - micronuclei
- FISH
Cytogenetic method - classic:
Metaphase analysis of dividing cells:
human lymphocytes, cell lines, fibroblasts, bone marrow cells
of experimental animal …
Human studies: examination of human lymphocytes
exposed to mutagene in vitro or lymphocytes of exposed
persons
– cultivation only 48 h – only 1st mitosis are examined
Structural CHA
intrachromosomal:
terminal deletion
acentric ring
interstitial deletion
centric ring
inversions:
pericentric
paracentric
interchromosomal:
symetric:
reciprocal translocation
chromatid exchange
asymetric:
dicentric chromosome
Chromatid aberrations (one chromatid):
chromatid break
Chromatid exchange
Chromosome aberrations (both chromatids)::
Chromosome break and ring
Chromosome exchanges
reciprocal translocation and dicentrics
Chromatid aberrations: chromatid break, chromatid
exchange are typical aberrations after action of
chemical mutagenes
Chromosome aberrations (on both chromatids):
chromosome break, terminal and interstitial
deletions, translocations, rings and dicentric
chromosomes are typical aberrations after ionizing
radiation
Chromatid exchange
Dicentric chromosome + difragment
Chromatid breaks
Cytogenetic method = biomarker of genotoxic
exposure !!!
= biomarker
of effect on human health (predictive for
cancer risk)
It is used for testing of exposed groups or testing of
exposed individuals
Analysis of acquired CHA:
group =  20 persons -100 of cells/person is analysed
individuals or group of  20 persons – 200 cells/person is analysed
% of aberrant cells, % of aberrations and types of aberrations are
evaluated
0-2% - normal level of aberrant cells
2-4% = increased level
4% = high level
Sister chromatid exchanges - SCE
+ BrdU (bromdeoxyuridine) for 2 cycles of division
BrdU = thymidine analogue
1.
2.
First mitosis:
BrdU is introduced to one DNA strand (new strand) of
both chromatids during replication → dark colour of both
chromatids
Second mitosis:
One chromatid is dark, second (with both strands
substituted by BrdU) is pale - delayed spiralization =>
pale staining
Observation of sister chromatid exchanges = method of
testing of mutagenic effects of environmental factors
Also proof of semiconservative DNA replication
Analysis of sister chromatid exchanges (SCE) =
method of mutagenicity testing
Mutagenes and carcinogenes increase frequency of SCE per cell
Detection of SCE in 30-50 cells and calculation of average
number of SCE per cell
SCEs in control
sample, not increased
Increased number
of SCEs
FISH in genotoxicology
Painting probes
analysis of translocations and other rearrangements –
2 painting probes are used
Exchanges of two painted chromosomes with other chromosomes are
scored and result is corrected for the whole genom  good agreement
of FISH with banding methods
Significance of FISH in genotoxicology:
- new knowledge about frequency and mechanisms of CHA
- FISH = quick method, easy scoring, many cells can be scored
- Detection of translocations – used for biological (retrospective)
dosimetry of radiation exposure – in case of long interval
between radiation accident and examination (translocations
are stable aberrations)
Dicentrics are unstable and suitable for biodosimetry
in short term after radiation
Biodosimetry – frequency of dicentrics or translocations increases
with radiation dose
Interphase analysis of CHA
Micronucleus test (MN)
Micronucleus = chromosomal fragment (mutagenic origin)
= whole chromosome - lost by anaphase lag
Analysis of human lymphocytes - analysed cell must go throug cell division
Method of cytokinesis block by cytochalasine (CB) binuclear cells –
micronucleus = small body stained darkly as nucleus
or detection of MN in bone marrow cell of experimental animals
Positive correlation of MN numbers with age, more MN in women
(probably part of MN is formed by chromosome X - lost in older women 1,4xmore MN in women)
Automatic analysis: flow cytometry
Micronucleus in
binuclear cell
http://dl1.cuni.cz/course/view.php?id=324 presentation
http://dl1.cuni.cz/course/view.php?id=324 supplementary text
to cytogenetics
Thompson &Thompson: Genetics in medicine, 7th ed.
Chapter 5: Principles of clinical cytogenetics: Introduction to
cytogenetics
Chapter 15: Prenatal diagnosis
+ informations from presentation