Methods of chromosome Methods of chromosome analysisanalysis

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

Knows few words

Inadequate reaction

Abnormal face

Short hands

, Heart abnormalities kidneyabnormalities

Plurimapformative Plurimapformative syndromesyndrome

What is his karyotype?What is his karyotype?

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

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

Thermostat

37º C

72hours

Bloodcells

+ growth broth

Cells indifferen t stages of cellcycle

Proliferation

After 72hours

3. Preparation of chromosomes for analysis

Cells indifferen t stages of cell

cycle

Hypotonicsolution

Cytostatic-

colchicine

Mitosisarresting

Cellsedimentatio

n

Chromosome dispersing

Slide with fixed cellsSlide with fixed cells Slide with metaphase platesSlide with metaphase plates

PaintingPainting

4. Painting of chromosomes4. Painting of chromosomes

5. Karyotype analysis5. Karyotype analysis

Printing ofpicture

Cutting individual

chromosomes

Preparation ofkaryotype

47,XY,+21 –Down syndrom47,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 karyotypeAnalysis of human karyotype

Metaphase chromosomes Prometapfase chromosomes Interphase chromosomes

Uniform painting, , , , G Q R C T banding, G R banding X and Y sex chromatin analysis Molecular citogenetical methods

, FISH mFISHSKYCGH

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

13

Type of bandingType of banding Used dyeUsed dye

GG Giemsa Giemsa

QQ QuinacrinQuinacrin(fluorescent) (fluorescent)

RR ((reversrevers) ) Giemsa or Giemsa or fluorescentfluorescent

CC (centromere) (centromere) Giemsa or Giemsa or fluorescentfluorescent

TT (telomere) (telomere) Giemsa or Giemsa or fluorescentfluorescent

Chromosome stainingChromosome staining

HomogenousHomogenous BandingBanding

GG QQ RR CC TT

Bands G+ = Q+ = R –Bands G+ = Q+ = R –

Bands G- = Q- = R +Bands G- = Q- = R +

Cum se colorează diferit acelaşi crs?Cum se colorează diferit acelaşi crs?

Uniform stainingUniform staining

C bandingC banding

Chromosomal bandingChromosomal banding

ChromosomeChromosome

BandBand

ArmArm

SubbandSubband

4p22.24p22.2

5q13.45q13.4

9p21.39p21.3

RegionRegion

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

1p35

1p22

1q23

1q41

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

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

25

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

Identification of 18 of 21 Identification of 18 and 21

FISH FISH in chromosomal in chromosomal analysisanalysis

46,XX46,XX 47,XX,+2147,XX,+21

47,XX,+2147,XX,+2147,XX,+2147,XX,+21

SKYSKY

mFISHmFISH

KaryotypeKaryotype

,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 2)2

NormalNormal Normal Normal polymorphismpolymorphism

ss

AneuploidyAneuploidy PolyploidyPolyploidy Chromosomal Chromosomal abberationsabberations

Abnormalphenotype

invtrob

Balanced structural aberrations

Fenotippatologic

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

– ,Monosomy 45 X

Aneuploidies

Abnormalphenotype

– Chrs D and G variations in ph or s :

, , , , – Chrs 1 9 16 9 Y variations in qh

Polymorphisms

Normalphenotype

,46 XX,46 XY

Normal karyotype

Fenotippatologic

deldupri

Unbalanced structural aberrations

??? Polymorphisms??? 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

Sex chromatin analysisSex chromatin analysis

X chromatin analysis = Barr body test – X chromatin analysis = Barr body test – identification of non-active X, in interphase nuclei of somatic cells

Y chromatin analysis = F body test – Y chromatin analysis = F body test – identification of 2/3 of qY identification of 2/3 of qY in interphase nuclei of somatic cells or spermsin interphase nuclei of somatic cells or sperms

Normal karyotype

- :Y chromosome 397 genes

• regulatory masculinisationgenes

•pseudogenes

•!!! q arm contains constitutive heterochromatin

X chromosome - : 1606 genes

• Structural somatic genes

• Regulatory feminisation;genes

• Structural feminisation; genes

• Structural masculinisationgenes

!!! Important!!! 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?How to explain this?

•Sexual dimorphism - balancedSexual dimorphism - balanced• Primary sexual traits - differentPrimary sexual traits - different• Secondary sexual traits - differentSecondary sexual traits - different• Somatic traits - similarSomatic traits - similar

45,X45,X 47,XXY47,XXY

46,XX46,XX 46,XY46,XY

- Active Xeuchromatin

- Active Xeuchromatin

Y chromosome

– p arm active

- q arminactive

- Inactive Xheterochromatin

F body

∅ = , 0 25 μm

Barr body

∅ = 1μm

Barr body – X sex chromatinBarr body – X sex chromatin

NrNrBarr Barr = X - 1= X - 1

Barr body test

XXX X XXXX XXXXX

Y YY

F body test

48,XXXY48,XXXY 45,X45,X 50,XXXXYY50,XXXXYY 49,XXXXX49,XXXXX

Barr body testBarr body test : X sexual chromatin

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

- represents a Barr body 1µ ;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 body, – 46 XY 0, – 47 XXX 2, - 47 XXY 1, – 45 X 0, – 48 XXXX 3

!!! . ( ) - M Lyon 1961 explanation of Barr body

Mary Lyon hypothesisMary Lyon hypothesis

After 16th day

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

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

,46 XX

, + 45 X active X paternalinactive

, + 45 X active X maternalinactive

F body testF body test : Y 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 25µ ;m in diameter- F body test is useful for prenatal identification of

: gender = No Y No F bodies

, – 46 XX 0, – 46 XY 1, – 47 XYY 2, - 47 XXY 1, – 48 XXYY 2, , ( ) – 46 X i Yp 0, , ( ) – ( ,46 X i Yq 1 0 5µ )m