Human

chromosomes

Human chromosomes
morphologic substrate of H and V;
supramolecular level of organization of genetic material (DNA +
histones + non-histones + RNA)

dynamic structures with different shape,

level of condensation, gene activity:
single-chromatid or two-chromatid;
chromatin or chromosome;
transcriptional active or inactive.
2

self-reproduction of chromosomes takes place

during S phase of interphase (replication)
chromosomes represent linkage groups of genes:
each chrs contains a specific number of genes;
each gene has a specific place in chrs - locus;
genes of one chromosome are inherited together

a diploid set of chromosomes is called karyotype:

23 pairs: 22 pairs of autosomes + 1 pair of gonosomes (XX or
XY)
Pair of chromosomes = homolougus chromosomes
3

Origin of gonosomes

landmarks of karyotype:

relative and absolute length of chrs

position of centromere = primary constriction - c
presence of secondary constrictions - h
presence of satellites - s
chromosomes clould be analyzed during:

metaphase (homogenous painting or banding)

prometaphase (banding)
interphase (hybridization with fluorescent probes)

chromosomes
have
heterogeneous
structure:

coding and non-coding sequences;

euchromatin and heterochromatin,
single copy and repetitive sequence;
GC and AT reach sequences;
transcribed and non-transcribed
sequences;
sequences associated with basic and
basic proteins.
!!! This explains origin of
chromosomal bands

Chromosomal number and structural

abnormalities induce developmental
abnormalities - sundromes
6

Structure of metaphase chromosomes.

Rchromosomal landmarks
Satellite
Secondary
constriction

Centromere

Primary
constriction

Sister
chromatids

The shape of chromosome depends on the

position of centromere
7

p x100
CI p
q

Human karyotype

Classification of chromosomes
By length:

By shape:

By type:

- Large

- Metacentric

- Autosomes

- Medium

- Submetacentri
c

- Gonosomes

- Small

- Acrocentric

By presence of
other
landmarks:
- h on p arm
- h on q arm
- satellites

Groups:
A 1-3

E 16-18

B 4,5

F 19,20

C X, 6-12

G 21, 22,
Y

D 13-15

10

Human karyotype and

chromosomal formula
46,XX normal karyotype
46,XY normal karyotype
47,XXY gonosomal trisomy (Klinefelter sdr.)
45,X gonosomal monosomy (Turner sdr.)
47,XY,+21 autosomal trisomy (Down sdr.)
45,XY,-21 autosomal monosomy (lethal)
46,XX,5p- - deletion in chrs. 5 (crie du chat)
11

Human karyotype and

chromosomal formula

46,XX,13ps- polymorphism
46,XY, 15ph++ polymorphism
46,XX,15qh+ abnormality
46,XY,16ph++ abnormality
46,XY,16qh++ polymorphism
46,XX,16q- abnormality
46,XX,16p+ abnormality
46,XX,13p- polymorphism
12

Methods of chromosome
analysis

What is his karyotype?

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

1. Collecting of nuclear cells (just leucocytes if

blood is used)

2. Cell culturing cells in different stages of cell cycle

Thermostat
37 C
Blood
cells
+
growth
broth

72
hours

Proliferation

After 72
hours

Cells in
differen
t stages
of cell
cycle

3. Preparation of chromosomes for analysis

Cytostatic
colchicine

Cells in
differen
t stages
of cell
cycle

Mitosis
arresting

Hypotonic
solution

Cell
sedimentati
on

Chromosom
e dispersing

4. Painting of chromosomes

Painting

Slide with fixed cells

Slide with metaphase plates

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

Metaphas
Prometapfas
Interphas
e
e
e
chromoso
chromosome
chromoso
mes
s
mes
X and Y
Molecular
G, R
Uniform G,Q,R,C,
sex
citogeneti
painting
T
banding
chromati
cal
banding
n
methods
analysis
FISH,
mFISH
SKY
CGH
*** No of bands per haploid set: 300-400 m / 550 pm /
850p

Type of banding Used dye

G

Giemsa

Quinacrin
(fluorescent)

R (revers)

Giemsa or
fluorescent

C (centromere)

Giemsa or
fluorescent

T (telomere)

Giemsa or
fluorescent
25

Chromosome staining
Homogenous

Banding

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

How the same chromosome is painted in

different patterns?

45,X

47,XX,+18

69,XXX

47,XXY

46,XX,5p-

47,XX,+13

47,XX,+21

Uniform staining

C banding

Chromosomal banding
Chromosome
Arm
Region
Band
Subband
4p22.2
5q13.4
9p21.3

1p35

1p22

1q23

1q41
46,XY,del(1)(p11p34)

46,XY,del(1)(p11p34)

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

FISH in chromosomal
analysis
Chromosome 4

Identification of X and 18 Identification of X, Y and 18

Identification of 18 of 21

Identification of 18 and 21
45

46,XX

47,XX,+21

47,XX,+21

47,XX,+21

SKY

mFISH

Karyotype
Normal

46,
XX
46,
XY

Normal
polymorphisms

Aneuploidy

46,XX,9qh
+

47,XX,
+21

46,XY,16q
h-

45,XY,12

46,XX,14s
++

48,XXX
Y
45,X

Polyploidy

Chromosomal
abberations

69,XX
X

46,XX,1q-

69,XX
Y

46,X,r(X)

46,XY,16p+
46,XY,del(5
p)
46,XX,t(12,
22)

Normal karyotype

46,XX
46,XY

Normal
phenoty
pe

Polymorphisms

Chrs D and G variations

in ph or s:
Chrs 1,9,16,9,Y
variations in qh
Trisomy 47,XXX; 47,XXY;
47,+21; 47,+13; 47,+18;
47,+8
Monosomy 45,X
inv
t
rob

Abnorm
al
phenoty
pe
Fenotip
patologi
c

del
dup
r
i

Fenotip
patologi
c

Aneuploidies

Balanced structural
aberrations

Unbalanced
structural
aberrations

Abnorm
al
phenoty
pe

??? 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 nonactive 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

X
chromosome
genes:

1606 Y chromosome - 397 genes:

Structural somatic genes

Regulatory
genes;
Structural
genes;

regulatory masculinisation
genes

feminisation pseudogenes
!!! q arm contains
feminisation constitutive heterochromatin

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?

Primary
sexual traits different
Sexual
Secondary
dimorphism sexual traits - balanced
different
Somatic traits
- similar

45,X

47,XXY

46,XX

Active X euchromatin

46,XY

Inactive X heterochromati
n

Active X euchromatin

Barr body
= 1m

Y
chromosome
p arm
active
q
arm inactive

F body
= 0,25

Barr body X sex chromatin

NrBarr = X - 1

48,XXXY

45,X

50,XXXXYY

49,XXXXX

Barr body test

XXX

XXXX

F body test

YY

XXXXX

Barr body test

X 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 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 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 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 0,25m 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 (0,5m)