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

Plurimapformative syndrome

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

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

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

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

Prometapfase chromosomes

Interphase chromosomes

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

13

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?

Uniform staining

C banding

Chromosomal banding

Chromosome

Band

Arm

Subband

4p22.2

5q13.4

9p21.3

Region

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 in chromosomal analysis

46,XX 47,XX,+21

47,XX,+2147,XX,+21

SKY

mFISH

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,XXY46,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

invtrob

Balanced structural aberrations

Fenotip patologic

Trisomy – 47,XXX; 47,XXY; 47,+21; 47,+13; 47,+18; 47,+8Monosomy – 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,XX46,XY

Normal karyotype

Fenotip patologic

deldupri

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

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 - 397 genes:

• regulatory masculinisation genes        

• pseudogenes

• !!! q arm contains constitutive heterochromatin

X chromosome - 1606 genes:

• Structural somatic genes

• Regulatory feminisation genes;

• Structural feminisation genes;

• Structural masculinisation genes

!!! 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

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

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 1m 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 body

Mary Lyon hypothesis

After 16th day

!!! In 46,XX cells – after 16th 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 0,25m 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 (0,5m)