Cy To Genetics

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.
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
by size and position of centromere for chromosomes of the same size.
3
The cell cycle
and the
detection of the
chromosomes
Human chromosomes are

examined in dividing cells
(bone marrow/placental cells,
lymphocytes).
The steps of chromosome assembly
1. chr.
(human)
DNA length 50
mm
chr. length 3-4

µm
10.000 x
condensation
The steps of chromosome assembly
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
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
46, XX
46, XY
10
Chromosome structure
11
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
• banding:: quinacrin staining (fluorescent)

Q-banding
Starting from the
centromere, the short and
the long chromosome
arms are numbered based
on the banding pattern.
12
Chromosome banding
G-banding Q-banding
R-banding C-banding T-banding

13
Chromosomal abnormalities
Human diseases resulting from altered kariotype are mostly caused by

ANEUPLOIDIES, TRANSLOCATIONS, and DELETIONS. 14
ANEUPLOIDY
ANEUPLOIDY is the abnormal number of chromosomes. The most frequent aneuploidia is trisomy,
having three instead of two chromosomes.
15
MITOTIC MEIOTIC
NON-DISJUNCTION NON-DISJUNCTION
16
MEIOTIC NON-DISJUNCTION—>
ANEUPLOIDY
17
MEIOTIC/MITOTIC NON-DISJUNCTION
18
Chromosome number abnormalities
Euploid chromosome mutation: Triploidity, Poliploid
Poliploidity
ity
Aneuploid chromosome mutation
Autosomal occurence Sex chromosomal occurence
Trisomy
21. trisomy 1/700 47, XXX Triplo- 1/1000 female
Down-syndrome X-syndrome
18. trisomy 1/13000 47, XXY 1/1000 male
Edwards- syndrome Klinefelter-
syndrome
13. trisomy 1/15000 47, XYY Dupla- 1/1000 male
Patau-syndrome Y-syndrome
Rare trisomies 3-,7-,8-,9-,12-
,14-,15-,19-,22-es
Monosomy 45, X0 Turner- 1/2500 female
syndrome
19
AUTOSOMAL ANEUPLOIDIES:
DOWN SYNDROME
20
DOWN SYNDROME
trisomy of the 21. chromosome 21
MATERNAL AGE & THE DOWN SYNDROME
22
EDWARDS SYNDROME
trisomy of the 18. chromosome
23
SEX CHROMOSOMAL ANEUPLOIDIES:
24
TURNER SYNDROME
25
Karyotype: 45X0
KLINEFELTER SYNDROME
26
Karyotype: 47XXY
TRIPLE X SYNDROME
27
Karyotype: 47XXX
SEX CHROMOSOMAL
ANEUPLOIDIES:YY SYNDROME
28
Karyotype: 47XYY
CLASSIFICATION OF STRUCTURAL CHROMOSOME
ABNORMALITIES BASED ON THE NUMBER OF
CHROMOSOMAL BREAK POINTS
Structural szerkezeti
abberation of chromosomes
kromoszóma aberráció
11 break
törés 2 breaks
2 törés 3 breaks
3 törés
On 22different
különböző
kromoszómán
chromosomes
terminális
Terminaldeléció
deletion 2On
különböző kromoszómán
2 different chromosomes On same
uazon chromosome
a kromoszómán inszerció
Insertion
Reciprocal
reciprok transzlokáció centrikus
Central fúzió
fusion uazon a kromoszóma
On same chromosome ellentétes
On kromoszóma
opposite
translocation vagy armkaron karon
chromosome arm
or
Robertson-féle
Robertson
transzlokáció
translocation
paracentrikus inverzió
Paracentric gyűrűRing
kromoszóma pericentrikus
Pericentric inverzió
inversion chromosome inversion
29
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
30
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
31
RING CHROMOSOME
 forms after telomere

t deletion
32
INVERSION
Paracentric inversion Pericentrikus inversion

33
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
34
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 Chronic myeloid leukemia

chromosome)
8-14 chromosome Burkitt - lymphoma
8-21 chromosome acute myeloid leukemia
35
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) fra(X)36 Y
FRAGILE X SYNDROME
 A mutation in the FMR-

FMR-1 gene (Xq28)
Xq28)
 CGG trinucleotid-
trinucleotid-repeat expansion
 5’ UTR affected
 repeat expansion is followed by methylation and inhibited expression
 FMR
FMR--1 protein is a RNA-
RNA-binding protein
 For the mental retardation the mGluR5 (metabotr
metabotropic
opic glutamate receptor)
receptor) might be
37
responsible
FRAGILE X SYNDROME
5-50 repeat
FMR-
FMR-1 gene
CGG healthy
50--200 repeat
50
‘Pre--mutation’
‘Pre CGG 200 - repeat
‘full mutation’ CGG

38
FISH: FLUORESCENT IN SITU
HIBRIDISATION
39
FISH
40
Direct and indirect method
Fluorescence
Excitation light
Fluorescencently
labeled antibody
Fluorescently labeled Non-fluorescently (DIG)

Non-
probe labeled probe
Detectable denaturated DNA

41
FISH
42
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
43
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
44
sex chromosome identification with painting probes.

two X chromosome (left) in a female cell, a Y chromosome (right) in a male cell.
45
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

46
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 Yp11.3
• X-linked ichthyosis Xp22.3
• Retinoblastoma (Rb-
(Rb-gene) 13q14
• Smith--Magenis syndrome
Smith 17p11.2
• Miller--Dieker syndrome
Miller 17p13.3
47
Medical application of FISH
21 trisomy: Down-syndrome 48
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 49

leukaemia cells.
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.
50
A BCR gene is on the 22.,the ABL gene is on the 9. chromosome. Translocation results
in fusion of the two genes.
51
Telomer specific FISH
52
53
X-CHROMOSOME INACTIVATION
Barr Body rule: #BB = #X-1

DOSAGE COMPENSATION
Heterochromatinization:
Xist RNA
DNA methylation
XXX:
Histone methylation Two Barr Bodies
RANDOM
DURING EARLY EMBRYOGENESIS
IRREVERSIBLE
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
X-CHROMOSOME INACTIVATION-
INACTIVATION-MO
MOS
SAI
AICISM
CISM
57
Anhidrotic Ectodermal Dysplasia
identify the following genotypes
58
Turner syndrome (XO) 59
Down syndrome (21 trisomy)
60
Klinefelter syndrome (XXY)
61
Normal woman (XX)
62
Normal man (XY)63

Cy To Genetics

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Cytogenetics

The goal of cytogenetics:

2. localisation of any (often abnormal)

Chromosome: condensed DNA with proteins attached (M phase of the

Human cells contain 46 chromosome, 44 autosome and 2 sex

A karyotype is the number and appearance of chromosomes in the

The chromosomes are depicted (by rearranging a microphotograph) in a

Human chromosomes are

chr. length 3-4

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

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

• G-banding: Giemsa staining

• R-banding: (reverse) modified Giemsa staining

• C-banding: centromer specific staining

• T-banding: telomer specific staining

• banding:: quinacrin staining (fluorescent)

R-banding C-banding T-banding

Human diseases resulting from altered kariotype are mostly caused by

trisomy of the 21. chromosome 21

trisomy of the 18. chromosome

 A piece of the chromosome is lost

 Terminal deletion - telomer is lost too – severe symptoms

Reciprocal translocation: breakpoints

breakpoints inside genes - genes with altered function

 forms after telomere

Paracentric inversion Pericentrikus inversion

5p Mental retardation, cardiac dev. problems

4-20. chromosome mental retardation, deformed face

X - 13. chromosome mental retardation

ACQUIRED TRANSLOCATIONS IN TUMORS

9-22 chr. (Philadelphia Chronic myeloid leukemia

8-21 chromosome acute myeloid leukemia

 most frequent inherited

 Xq27.3 –increased fragility

 A mutation in the FMR-

‘full mutation’ CGG

Fluorescently labeled Non-fluorescently (DIG)

Detectable denaturated DNA

sex chromosome identification with painting probes.

RB1 gene on the 13. chromosome

gene specific FISH

The p53 tumorsuppressor gene shown by gene specific FISH probe in 49

The Philadelphia chromosome is frequently occuring in chronic myeloid leukaemia (CML).

Barr Body rule: #BB = #X-1

THERE ARE GENES EXPRESSED ON THE INACTIVE X

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