Dr. Nasser A ElhawaryProfessor of Medical Genetics

A chromosome distincts into 2 “sister chromatides”.

They are linked together via a centromere

2 Chromatides

Centromere

Human metaphase

Human Chromosomes…

Contains DNA and Protein 46 chromosomes Autosomes: Pairs 1-22 Sex chromosmes

Tjio & Levan reported it is 46 chrom. in 1956

The chr’s are present in pairs ‘homologs’ (one from female, the other from male).

Diploid cells (2n): Cells that contain pairs of homologous chr’s.

Haploid cells (n): Certain cells (sperm/egg gametes) contain only one copy of each chr.

At fertilization, the fusion of haploid gametes together produces a cell carrying the diploid no. of chr’s (zygote).

Identification of Chromosomes…

Classification of Chromosomes

The centromere (1ry constriction) divides the chr into 2 arms (petit short ‘p’-arm & long ‘q’-arm).

The location of centromere in each chr is characteristic for a given chromosome.

‘Metacentric’: a chromosome with a centrally placed centromere.

‘Submetacentric’: a chromosome with a centromere close to one end than the other.

‘Acrocentric’: a chr with the centromer placed very close to one end.

Classification of Chromosomes…

Human chromosomes are arranged in groups from A to G.

Each group is defined by chromosomal size and centromere location.

In 1960s, new staining procedures were developed that resulted in banded chromosomes (‘dark’ and ‘light’).

Banding protocols: G-banding, R-banding, Q-banding, C-banding, etc.

Chromosome Banding…

Chromosome Banding…

A

karyptype

of

G-banded

Human

Chromosomes

R-banding…

Q-banding

C-banding…

What is the importance of high resolution banding?

Identification of Chromosomes…

Each arm is subdivided into numbered regions starting at the centromere.

Thus, any region can be identified by a descriptive address such as Xq27.3/

FMR1 gene

Karyotype Analysis…

The banding

pattern of the

chromosomes

in the human

karyotype.

Banding chromosomes…

G-Banding using trypsin and Giemsa stain the chromatin in 2 main forms:

Euchromatin: stains ‘light’ and consists of genes which are actively expressed.

Heterochromatin stains ‘dark’ and is made up largely of inactive unexpressed repetitive DNA.

Molecular Cytogenetics

Fluorescence In-Situ Hybridization (FISH):

It is ability of a portion of ssDNA

(i.e. probe) to anneal with its

complement-ary target sequence

on: i) a metaphase chromosome,

ii) interphase nucleus or iii)

extended chromatin fiber.

FISH is widely used in clinical

diagnostic purposes.

In FISH, the DNA probe is labeled

with a fluorochrome + patient’s

chrom visualized using

fluorescent microscope.

FISH…

1- Centromeric probes.

2- Chromosome-specific unique-

sequence probes.

3- Telomeric probes.

4- Whole chromosome paint probes.

Types of FISH… 1- Centromeric probes

FISH of interphase nuclei with

Centromeric probes for

chromosomes 18.X and Y:

showing 3 signals consistent

with trisomy 18.

2- Unique sequence probes

are useful for identifying tiny

submicroscopic deletions &

duplications.

Also, use of interphase FISH

probe to identify HER2 over-

expression in breast tumors.

Metaphase image: Chromosome band 7q11.23 showed deletion associated with Williams syndrome. Normal chrom. has 2 signals (green) for the control probe and the ELN gene probe signal (orange), but the deleted chrom. shows only the control probe signal

Telomeric probes have been used for

identifying tiny ‘cryptic’ subtelomeric

abnormalities, e.g. deletions,

translocations.

Types of FISH… Telomeric probes

These consists of a cocktail of probes obtained

from different parts of a particular chromosome.

When this mixture of probes is used together in

a single hybridization, the entire chromosome

fluoresces (i.e. is “painted”).

Useful in rearrangements (e.g. translocations) &

additional chromosomal materials.

Types of FISH… 3- Whole-chrom paint probes

Types of FISH… Whole-chrom paint probes

Chrom painting showing a reciprocal translocation involving chrom 3 (red) & 20 (green).

M-FISH or Spectral karyotyping (SKY) uses pools of whole human chrom. paint probes to provide a multicolor human karyotype. Each homologous chrom. shows a unique color.

These are useful to detect chromosomal rearrangements (e.g. deletions, trans-locations), ring chrom, …

Types of FISH… Whole-chrom paint probes

Types of FISH… Whole-chrom paint probes

M-FISH showing complex chromosome rearrangement involving chromosomes 4, 8, 13, 18, and 21

• CGH was originally developed to overcome the difficulty of obtaining good-quality metaphase preparations from solid tumors.

• CGH enables the detection of regions of allele loss and gene amplification.

• Tumor ‘test’ DNA is labeled with green paint & control normal DNA with red paint.

• The two samples are mixed and hybridized com-petitively to normal metaphase chromosomes.

• watch the green-to-red ratio????

CGH Analysis showing areas of

gene amplification & reduction

(del) in tumor DNA.

DAPI: diamidophenylindole

FITC: Fluorescein isothiocyanate

• CGH extended to include the analysis of single cells for prenatal diagnosis following whole-genome amplification.

• CGH limits for more than 10Mb for losses and 2Mb for gains.

• Microarray or array CGH is likely to replace metaphase CGH.

CGH…

ChromosomalAbnormalities

NumericalAbnormalities

StructuralAbnormalities

Numerical Abnormalities

Aneuploidy: Loss or gain one or more chromosome

Polyploidy: Addition of one or more complete haploid sets (69, 92 chromosomes).

Monosomy: Loss of a single chromosome (Turner syndrome: 45,X0)

Trisomy: gain of a single chromosome (e.g. Down syndrome: 47,XY,+21).

Aneuploidy.. Numerical abnormality…

Down syndromeTrisomy 21

Turner syndromeMonosomy

A Karyotype of triploidy cell, 69,XXX

Mechanism of Trisomy

Structural Abnormalities

1.Translocation: Transfer a genetic material

from one chromosome to another.

2.Reciprocal translocation: Breakage in 2

chromosomes and exchanged.

3.Robertsonian translocation: Breakage

close to centromeres of acrocentric chrom.

(e.g. Down syndrome)

Translocation

Origin of translocationin mother giving Down syndrome

4- Deletions: loss of part of a chromosome (e.g. Wolf syndrome ‘4p-’, cri du chat syndrome ‘5p-’).

5- Insertions: a segment of one chromosome is inserted into another chromosome.

6- Inversions: a two-break rearrangement in a chrom. which re-inserted in the inverted positions.

7- Ring chromosome: 2 breaks leaving sticky ends which re-unit to give ring chromosome.

8- Isochromosomes: from loss of one arm of the chrom. with duplication of the other.

Structural abnormalities…

Cri du chat syndrome (5p–)http://en.wikipedia.org/wiki/Cri_du_chat

Insertion

Ring chromosome(1926)

A) Pericentric inversion

B) Paracentric inversion

Inversion:

Mosaicism & Chimerism

Mosaic: Presence of two or more cell lines in an

individual, but derived from the same zygote.

Mosaic produces from non-disjunction in an

early embryonic mitotic division (mosaic trisomy

21).

Chimerism: Presence of two or more cell lines

in an individual, derived from more than one

zygote.

Blood Chimeras & Dispermic chimeras.

Mosaicism & Chimersim mechanism