Download Chromosome

Cytogenetics
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Cytogenetics
Cytogenetics is the study of
chromosomes and their role in
heredity.
The goal of cytogenetics:
1. diagnosis of chromosomal
abnormalities.
2. localisation of any (often abnormal)
chromosomal region/DNA sequence.
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Basic Cytogenetic Terms
Chromatin: non condensed DNA with proteins attached (interphase of
the cell cycle)
Chromosome: condensed DNA with proteins attached (M phase of the
cell cycle)
Human cells contain 46 chromosome, 44 autosome and 2 sex
chromosome.
A karyotype is the number and appearance of chromosomes in the
nucleus of a eukaryotic cell. The term is also used for the complete set
of chromosomes in a species, or an individual organism.
The chromosomes are depicted (by rearranging a microphotograph) in a
standard format known as a karyogram or idiogram: in pairs, ordered
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by size and position of centromere for chromosomes of the same size.
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The cell cycle
and the
detection of the
chromosomes
Human chromosomes are
examined in dividing cells
(bone marrow/placental cells,
lymphocytes).
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The steps of chromosome assembly
1. chr.
(human)
DNA length 50
mm
chr. length 3-4
µm
10.000 x
condensation
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The steps of chromosome assembly
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The history of human chromosome
identification
1879. Arnold: First visualization of human chromosomes.
1888. Waldeyer: The word chromosome (chroma: color, soma: body)
1882. Walther Flemming: 20-28 chromosomes in cells of cornea
1921. T.S. Painter: 48 human chromosomes, X & Y chromosomes (Science)
1956. Jo Hin Tijo és Albert Levan : 46 human chromosomes (Hereditas)
1959. Lejeune: trisomy 21=Down syndrome
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The history of human chromosome
identification
Kariotyping conferences
1960. Denver: chromosomess numbered (1-22) based on their size
1963. London: chromosome grouping (A-G)
1966. Chicago: big chromosome syndromes
1971. Paris, 1976. Mexico, 1978. Stockholm: chromosome banding
1995. ISCN : International System for Human Cytogenetic Nomenclature
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46, XX
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46, XY
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Chromosome structure
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Chromosome banding
The chromosome banding techniques use different chemicals
to stain the chromosomes in order to identify them or to show
chromosomal rearrangements. Based on the stains used the
following banding techniques exist:
•
G-banding: Giemsa staining
•
R-banding: (reverse) modified Giemsa staining
•
C-banding: centromer specific staining
•
T-banding: telomer specific staining
•
Q-banding
banding:: quinacrin staining (fluorescent)
Starting from the
centromere, the short and
the long chromosome
arms are numbered based
on the banding pattern.
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Chromosome banding
G-banding
R-banding
Q-banding
C-banding
T-banding
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Chromosomal abnormalities
Human diseases resulting from altered kariotype are mostly caused by
ANEUPLOIDIES, TRANSLOCATIONS, and DELETIONS.
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ANEUPLOIDY
ANEUPLOIDY is the abnormal number of chromosomes. The most frequent aneuploidia is trisomy,
having three instead of two chromosomes.
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MITOTIC
NON-DISJUNCTION
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MEIOTIC
NON-DISJUNCTION
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MEIOTIC NON-DISJUNCTION—>
ANEUPLOIDY
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MEIOTIC/MITOTIC NON-DISJUNCTION
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Chromosome number abnormalities
Euploid chromosome mutation: Triploidity, Poliploid
Poliploidity
ity
Aneuploid chromosome mutation
occurence
Sex chromosomal occurence
Autosomal
Trisomy
21. trisomy
Down-syndrome
1/700
47, XXX TriploX-syndrome
1/1000 female
18. trisomy
Edwards- syndrome
1/13000
47, XXY
Klinefeltersyndrome
1/1000 male
13. trisomy
Patau-syndrome
1/15000
47, XYY DuplaY-syndrome
1/1000 male
45, X0 Turnersyndrome
1/2500 female
Rare trisomies 3-,7-,8-,9-,12,14-,15-,19-,22-es
Monosomy
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AUTOSOMAL ANEUPLOIDIES:
DOWN SYNDROME
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AUTOSOMAL ANEUPLOIDIES:
DOWN SYNDROME
trisomy of the 21. chromosome
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MATERNAL AGE & THE DOWN SYNDROME
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AUTOSOMAL ANEUPLOIDIES:
EDWARDS SYNDROME
trisomy of the 18. chromosome
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SEX CHROMOSOMAL ANEUPLOIDIES:
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SEX CHROMOSOMAL ANEUPLOIDIES:
TURNER SYNDROME
Karyotype: 45X0
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SEX CHROMOSOMAL ANEUPLOIDIES:
KLINEFELTER SYNDROME
Karyotype: 47XXY
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SEX CHROMOSOMAL ANEUPLOIDIES:
TRIPLE X SYNDROME
Karyotype: 47XXX
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SEX CHROMOSOMAL
ANEUPLOIDIES:YY SYNDROME
Karyotype: 47XYY
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CLASSIFICATION OF STRUCTURAL CHROMOSOME
ABNORMALITIES BASED ON THE NUMBER OF
CHROMOSOMAL BREAK POINTS
Structural szerkezeti
abberation
of chromosomes
kromoszóma
aberráció
11 break
törés
3 breaks
3 törés
2 breaks
2 törés
különböző
On 22different
kromoszómán
chromosomes
terminális
Terminaldeléció
deletion
On
same
chromosome
uazon
a kromoszómán
2On
különböző
kromoszómán
2 different
chromosomes
Reciprocal
reciprok transzlokáció
translocation
centrikus
fúzió
Central
fusion
vagy
Robertson-féle
Robertson
transzlokáció
or
uazon
a kromoszóma
On same
chromosome
karon
arm
inszerció
Insertion
ellentétes
kromoszóma
On
opposite
karon
chromosome
arm
translocation
Paracentric
paracentrikus inverzió
inversion
gyűrűRing
kromoszóma
chromosome
pericentrikus
Pericentric inverzió
inversion
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DELETION
 A piece of the chromosome is lost
 Deletion mapping
 Intersticial deletion -
Prader-Willi; Angelman syndromes del 5(q11-13)
Williams syndrome
 Terminal deletion - telomer is lost too – severe symptoms
Cri du chat syndrome
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TRANSLOCATION
Reciprocal translocation: breakpoints
are on 2 homologous or on 2 different
chromosomes
balanced translocation
break points are mostly in non coding regions
breakpoints inside genes
- genes with altered function
- genes with altered activity
- genes with altered expression
- genes with loss of function
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RING CHROMOSOME
 forms after telomere
t
deletion
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INVERSION
Paracentric inversion
Pericentrikus inversion
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INHERITED DELETIONS
DELETION
SYMPTOM
5p
Mental retardation, cardiac dev. problems
18p
18
p
Physical and mental retardation
Xq (Fragil
(Fragile
e X)
Deformed face, autism
22q
22
q
Thyroid gland/ parathyroid gland development
defect
13q
13
q
Tumors, retinoblastoma
INHERITED TRANSLOCATIONS
TRANSLOCATION
SYMPTOM
4-20. chromosome
mental retardation, deformed face
X - 13. chromosome
mental retardation
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ACQUIRED DELETIONS IN TUMORS
DELETION
TUMOR
APC--gene
APC
colorectal
Rb (Retinoblastoma) gene
any tumor
P53 gene
any tumor
22. chromosome
acute myeloid leukemia
ACQUIRED TRANSLOCATIONS IN TUMORS
TRANSLOCATION
TUMOR
9-22 chr. (Philadelphia
chromosome)
Chronic myeloid leukemia
8-14 chromosome
Burkitt - lymphoma
8-21 chromosome
acute myeloid leukemia
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FRAGILE X SYNDROME
 most frequent inherited
mental retardation
 symptoms:
- big head, elongated
face, big ears
- mild – severe mental
retardation
 Xq27.3 –increased fragility
Xq27.3
X fra(X)
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fra(X)36 Y
FRAGILE X SYNDROME
 A mutation in the FMRFMR-1 gene (Xq28)
Xq28)
 CGG trinucleotidtrinucleotid-repeat expansion
 5’ UTR affected
 repeat expansion is followed by methylation and inhibited expression
 FMR
FMR--1 protein is a RNARNA-binding protein
 For the mental retardation the mGluR5 (metabotr
metabotropic
opic glutamate receptor)
receptor) might be
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responsible
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FRAGILE X SYNDROME
5-50 repeat
FMRFMR
-1 gene
healthy
CGG
50--200 repeat
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‘Pre--mutation’
‘Pre
CGG
‘full mutation’
200 - repeat
CGG
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FISH: FLUORESCENT IN SITU
HIBRIDISATION
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FISH
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Direct and indirect method
Fluorescence
Excitation light
Fluorescencently
labeled antibody
Fluorescently labeled
probe
Non-fluorescently (DIG)
Nonlabeled probe
Detectable denaturated DNA
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FISH
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Types
of
FISH
probes
chromosome specific probes: for detection of chromosome number abnormalities
X (red) and Y (green) chromosome specific probes in healthy male and female cells
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Types of FISH probes
whole chromosomes are highlighted with the painting probes – good for detecting translocations
1. chromosome (left), 22. chromosome (middle), 3. chromosome (right) shown with painting probes
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Types of FISH probes
sex chromosome identification with painting probes.
two X chromosome (left) in a female cell, a Y chromosome (right) in a male cell.
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Types of FISH probes
the locus/gene specific probes are used to detect any gene of interest, frequently absence or
amplification of tumor suppressors of oncogenes
RB1 gene on the 13. chromosome
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Locus/gene specific FISH probes
•
Prader--Willi syndrome
Prader
15q11-13
15q11-
•
Angelman syndrome
15q11--13
15q11
•
Di--George syndrome /VCFS
Di
22q11.2
•
Williams syndrome
7q11.23
•
Wolf--Hirschhorn syndrome
Wolf
4p16.3
•
Cri du Chat syndrome
Xp22.3
•
SRY gene
•
X-linked ichthyosis
Xp22.3
•
Retinoblastoma (Rb(Rb-gene)
13q14
•
Smith--Magenis syndrome
Smith
17p11.2
•
Miller--Dieker syndrome
Miller
17p13.3
Yp11.3
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Medical application of FISH
21 trisomy: Down-syndrome
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Medical application of FISH
gene specific FISH
probes proved
amplification of
oncogenes (erb-B2,
EGFR, myc) and
deletion of tumor
supressor genes
(p53, Rb) in a vast
range of tumors
The p53 tumorsuppressor gene shown by gene specific FISH probe in
leukaemia cells.
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Medical application of FISH
Philadelphia chromosome
(Ph1)
abl (Abelson cluster region) gene–
encodes a tirosin kinase
bcr (breakpoint cluster region)
The Philadelphia chromosome is frequently occuring in chronic myeloid leukaemia (CML).
Diagnosis is based on gene specific FISH probes.
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Medical application of FISH
A BCR gene is on the 22.,the ABL gene is on the 9. chromosome. Translocation results
in fusion of the two genes.
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Telomer specific FISH
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X-CHROMOSOME INACTIVATION
Barr Body rule: #BB = #X-1
DOSAGE COMPENSATION
Heterochromatinization:
Xist RNA
DNA methylation
Histone methylation
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XXX:
Two Barr Bodies
X-CHROMOSOME INACTIVATION
RANDOM
DURING EARLY EMBRYOGENESIS
IRREVERSIBLE
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X-CHROMOSOME INACTIVATION
XX
XO
?
THERE ARE GENES EXPRESSED ON THE INACTIVE X
CHROMOSOME!
THE ROLE OF PSEUDOAUTOSOMAL REGIONS (PRESENT ON
BOTH X & Y):
CHIASMATA FORMATION, TO ENSURE PROPER SEGREGATION
OF X & Y CHROMOSOMES
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X-CHROMOSOME INACTIVATIONINACTIVATION-MO
MOS
SAI
AICISM
CISM
Anhidrotic Ectodermal Dysplasia
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identify the following genotypes
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Turner syndrome (XO)
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Down syndrome (21 trisomy)
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Klinefelter syndrome (XXY)
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Normal woman (XX)
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Normal man (XY)63
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