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Dr. Nasser A ElhawaryProfessor of Medical Genetics
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A chromosome distincts into 2 “sister chromatides”.
They are linked together via a centromere
2 Chromatides
Centromere
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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
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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…
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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.
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Classification of Chromosomes…
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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…
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Chromosome Banding…
A
karyptype
of
G-banded
Human
Chromosomes
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R-banding…
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Q-banding
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C-banding…
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What is the importance of high resolution banding?
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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
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Karyotype Analysis…
The banding
pattern of the
chromosomes
in the human
karyotype.
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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.
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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.
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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…
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1- Centromeric probes.
2- Chromosome-specific unique-
sequence probes.
3- Telomeric probes.
4- Whole chromosome paint probes.
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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.
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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
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Telomeric probes have been used for
identifying tiny ‘cryptic’ subtelomeric
abnormalities, e.g. deletions,
translocations.
Types of FISH… Telomeric probes
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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
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Types of FISH… Whole-chrom paint probes
Chrom painting showing a reciprocal translocation involving chrom 3 (red) & 20 (green).
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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
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Types of FISH… Whole-chrom paint probes
M-FISH showing complex chromosome rearrangement involving chromosomes 4, 8, 13, 18, and 21
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• 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????
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CGH Analysis showing areas of
gene amplification & reduction
(del) in tumor DNA.
DAPI: diamidophenylindole
FITC: Fluorescein isothiocyanate
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• 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…
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ChromosomalAbnormalities
NumericalAbnormalities
StructuralAbnormalities
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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).
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Aneuploidy.. Numerical abnormality…
Down syndromeTrisomy 21
Turner syndromeMonosomy
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A Karyotype of triploidy cell, 69,XXX
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Mechanism of Trisomy
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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)
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Translocation
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Origin of translocationin mother giving Down syndrome
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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…
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Cri du chat syndrome (5p–)http://en.wikipedia.org/wiki/Cri_du_chat
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Insertion
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Ring chromosome(1926)
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A) Pericentric inversion
B) Paracentric inversion
Inversion:
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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.
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Mosaicism & Chimersim mechanism