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Methods of chromosome

analysis

He is 4 years old (looks like 2 y.o.)

Knows few words

Inadequate reaction

Abnormal face

Short hands

Heart abnormalities, kidney abnormalities

Plurimapformativesyndrome

What is his karyotype?

1. Collecting of nuclear cells (just leucocytes if b lood is used)

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2. Cell culturing – cells in different stages of cell cycle

Thermostat

37º C

72 hours

Blood cells

+ growth broth

Cells in different stages of cell cycle

Proliferation

After 72 hours

3. Preparation of chromosomes for analysis

Cells in different stages of cell cycle

Hypotonic solution

Cytostatic -colchicine

Mitosis arresting

Cell sedimentation

Chromosome dispersing

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Slide with fixed cells Slide with metaphase plates

Painting

4. Painting of chromosomes

5. Karyotype analysis

Printing of picture Cutting individual chromosomes

Preparation of karyotype

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47,XY,+21 –Down syndrom

Cytogenetic methods used for

karyotype analysis:

To identify numeric aberrations – analysis of

metaphase chromosomes uniformly painted.

To identify numeric or structural aberrations – analysis

of metaphase or prometaphase chromosomes using Q,

G, R banding.

For exact identification of chromosomes – analysis of

metaphase chromosomes using T or C banding.

Analysis of human karyotype

Metaphase chromosomes

Prometapfasechromosomes

Interphasechromosomes

Uniform painting

G,Q,R,C,T banding

G, R banding

X and Y sex chromatin analysis

Molecular citogenetical

methodsFISH, mFISH

SKYCGH

*** No of bands per haploid set: 300-400 m / 550 pm / 850p

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Type of banding Used dye

G Giemsa

Q Quinacrin

(fluorescent)

R (revers) Giemsa or fluorescent

C (centromere) Giemsa or fluorescent

T (telomere) Giemsa or fluorescent

Chromosome staining

Homogenous Banding

G Q R C T

Bands G+ = Q+ = R –

Bands G- = Q- = R +

Cum se colorează diferit acelaşi crs?

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Uniform staining

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C banding

Chromosomal banding

Chromosome

Band

Arm

Subband

4p22.2

5q13.4

9p21.3

Region

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46,XY,del(1)(p11-p34)

1p35

1p22

1q23

1q41

46,XY,del(1)(p11-p34)

46,Y,del(X)(q12.1-q24.3)

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Chromosome 4 Identification of X and 18 Identification of X, Y and 18

Identification of 18 of 21 Identification of 18 and 21

FISH in chromosomal

analysis

46,XX 47,XX,+21

47,XX,+2147,XX,+21

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SKY

mFISH

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Karyotype

46,XX

46,XY

46,XX,9qh+

46,XY,16qh-

46,XX,14s++

47,XX,+21

45,XY,-12

48,XXXY

45,X

69,XXX

69,XXY

46,XX,1q-

46,XY,16p+

46,X,r(X)

46,XY,del(5p)

46,XX,t(12,22)

Normal Normal

polymorphisms

Aneuploidy Polyploidy Chromosomal

abberations

Abnormal phenotype

inv

t

rob

Balanced structural aberrations

Fenotip patologic

Trisomy – 47,XXX; 47,XXY; 47,+21; 47,+13; 47,+18; 47,+8

Monosomy – 45,X

Aneuploidies

Abnormal phenotype

Chrs D and G – variations in ph or s:

Chrs 1,9,16,9,Y – variations in qh

Polymorphisms

Normal phenotype

46,XX

46,XY

Normal karyotype

Fenotip patologic

deldup

r

i

Unbalanced structural aberrations

??? Polymorphisms

• p and q – contain coding and non-coding regions

• p+ or p- - abnormality • q+ or q- - abnormality

• c longer or shorter – polymorphism• t longer or shorter – polymorphism

• h+ or h- - polymorphism• s+ or s- - polymorphism

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Sex chromatin analysis

X chromatin analysis = Barr body test – identification of non-active

X, in interphase nuclei of somatic cells

Y chromatin analysis = F body test – identification of 2/3 of qY

in interphase nuclei of somatic cells or sperms

Normal karyotype

Y chromosome - 344 genes:

• regulatory masculinisation genes

• pseudogenes

• !!! q arm contains constitutive heterochromatin

X chromosome - 1529 genes:

•Structural somatic genes

•Regulatory feminisation genes;

•Structural feminisation genes;

•Structural masculinisation genes

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!!! Important

• X chromosome – is mandatory for and :

• In 46,XX – only one X is active

• In 46,XY – are active both X and Y

• In 47,XXX - only one X is active

• In 47, XXY- are active only one X and one Y

• In 48, XXXY- are active only one X and one Y

• Y chromosome – is mandatory for

How to explain this?

Sexual dimorphism - balanced

• Primary sexual traits - different

• Secondary sexual traits - different

• Somatic traits - similar

45,X 47,XXY

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46,XX 46,XY

Active X -euchromatin

Active X -euchromatin

Y chromosome

p arm – active q arm - inactive

Inactive X -heterochromatin

F body

= 0,25 μm

Barr body

= 1μm

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Barr body – X sex chromatin

NrBarr = X - 1

Barr body test

XXX X XXXX XXXXX

Y YY

F body test

48,XXXY 45,X 50,XXXXYY 49,XXXXX

Barr body testX sexual chromatin:

- represents an inactive X, facultative heterochromatin, in somatic cells 46,XX;

- represents a Barr body 1 m in diameter;

-Barr body test is useful for identification of No of X chromosomes in somatic cells: No X = No Barr + 1.46,XX – 1 Barr body46,XY – 0 47,XXX – 247,XXY - 145,X – 048,XXXX – 3!!! M. Lyon (1961) - explanation of Barr bod y

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Mary Lyon hypothesis

After 16th day

!!! In 46,XX cells – after 16 th day of development only one X remains active

In 50% of cells – maternal X is active, in 50% - paternal X

46,XX

45,X active +X paternal inactive

45,X active + X maternal inactive

F body testY sexual chromatin:

- represents 2/3 of q arm of Y chromosome, its a constitutive heterochromatin in somatic cells or 50% of sperms (23,Y only);

- represents fluorescent F body 0,25 m in diameter;

- F body test is useful for prenatal identification of gender: No Y = No F bodies

46,XX – 046,XY – 1 47,XYY – 247,XXY - 148,XXYY – 246,X,i(Yp) – 046,X,i(Yq) – 1 (0,5 m)