CHROMOSOMAL STRUCTURES AND CHROMOSOMAL MUTATIONS

The human genome consists of 2.9 billion nucleotide base pairs of DNA organized into 23 chromosomes.
Alterations of the DNA sequence may affect not only the phenotype of an individual but the progeny of that
individual as well. A transmissible (inheritable) change in the DNA sequence is a mutation or polymorphism.
DNA mutations can affect a single nucleotide or millions of nucleotides, even whole chromosomes, and thus
can be classified into three categories: gene, chromosome, and genome mutations.

Euploid
A cell or cell population with a normal complement of chromosomes
Aneuploid
Aneuploidy is mostly observed as increased numbers of chromosomes because
the loss of whole chromosomes are generally not compatible with survival.

An extended DNA double helix undergoes an 8,000-fold compaction to make a

metaphase chromosome. Chromosome topology (state of compaction of the DNA
double helix) affects gene activity.

CHROMOSOME MORPHOLOGY
The centromere consists of tandem repeats of 171 base pair
sequences flanking sets of single repeat units, or monomers repeated in
groups in a higher-order repeat array. The kinetochore is a protein
structure that connects centromere chromatin to spindle apparatus.

The arms of the Metacentric chromosome are of

equal size. Submetacentric chromosomes divide
the chromosomes into long arms and short
arms. Acrocentric centromere are very near the
ends the chromosomes

VISUALIZING CHROMOSOMES

Conventional cytological stains:

This allows unequivocal identification of every chromosome and the direct detection of some
chromosomal abnormalities: FEULGEN, WRIGHT, HEMATOXYLIN

Fluorescent dyes
Quinacrine and quinacrine mustard.

Chemical dye
Giemsa

Heterochromatin stains darkly by G or Q banding. Euchromatin stains

darkly by R banding. C banding stains centromere.
What are bands?
The alternating light and dark stained demarcations which appear along the length of each
chromosome. The banding patterns are specific for each chromosome pair.

Human chromosome could be identified by its characteristic banding pattern. Q-banding, Required fluorescent
microscope, not widely used. G bands, similar to those seen in Q banding. R bands can also be visualized
after staining with acridine orange. Alkali treatment of chromosomes results in centromere staining, or C
banding.

Staining – dyes bind to the DNA or protein of a chromosome and allow visualization by light microscopy.
Giemsa – routine stain

Special staining
● Q – banding / quinacrine flourescence staining – routine karyotyping but replaced with G-banding which
allow permanent stain preparation.
● Rapid identification of Y chromosome
● C- banding – to evaluate constitutive heterochromatin or to determine if the chromosomes has two
centromeres.
● Centromeres appear as single dark spot
● R- banding – the telomeres should stain as dark and their absence as the result of a deletion is more
● obvious.

KARYOTYPING
is the direct observation of metaphase chromosome structure by arranging metaphase chromosomes
according to size. It can also be used to detect chromosomal mutations such as translocations, which are the
exchange of genetic material between chromosomes.
Karyotype is the complete set of chromosomes in a cell.

SPECIMEN
● Routine studies – heparinized blood.
● Hematologic disorders – bone marrow samples
● Fibroblast cultures from skin biopsies
● Not routinely done due its invasive in nature
○ liver, kidney, lung, and muscles
● Amniotic fluid – prenatal analysis
○ done between 16 – 18 weeks of gestation (20-30ml)
●
● Chorionic villus sampling – tissues from the developing placenta. 10 - 14 weeks of gestation
● Cordocentesis or percutaneous umbilical cord sampling > 20 weeks of gestation

All samples
1. must be collected in a sterile manner.
2. Must be transported to the laboratory as soon as possible.
Blood, bone marrow, amniotic fluid and chorionic villi: room temperature
Solid tissues: wet ice
Cell Culture Technique
Bone marrow and blood
aliquoted directly in culture medium
Amniotic fluid cells, CV and solid tissues
grown as a monolayer in situ.
Amniotic fluid – centrifuge
5-10 days – typical culture timE, harvested

● Swelled hypotonically and are then fixed

● Automated robotic harvester has been developed

CHROMOSOMAL MUTATIONS: TRANSLOCATION

Types of translocation
1. Reciprocal - each chromosome breaks, and the broken chromosomes reassociate or recombine with one
another.
When this type of translocation results in no gain nor loss of chromosomal material = BALANCED
2. Robertsonian translocation - involves the movement of the long arm of an acrocentric chromosome to the
centromere of another acrocentric chromosome = UNBALANCED

Deletion
is a loss of chromosomal material. Large deletions
covering millions of base pairs can be detected using
karyotyping
Microdeletion is not usually detected.
Insertion
is a gain of chromosomal material.
Inversions
result from excision, flipping, and reconnecting chromo-
somal material within the same chromosome.
Pericen- tric inversions
include the centromere in the inverted region, whereas
paracentric inversions involve sequences within one arm of the chromosome. An iso- chromosome is a
metacentric chromosome that results from transverse splitting of the centromere during cell division.

● Ring chromosome results from deletion of genetic regions from

ends of the chromosome and a joining of the ends to form a ring.
● Derivative chromosome is an abnormal chromosome consisting
of translocated or otherwise rearranged parts from two or more
unidentified chromosomes joined to a normal chromosome.

Results of karyotyping analyses are expressed as the number of

chromosomes per nucleus (normal is 46), the type of sex chromosomes
(normal is XX or XY), followed by any genetic abnormalities observed. A
normal karyotype is 46,XX in a female or 46,XY in a male.
FLUORESCENCE IN SITU HYBRIDIZATION
● is a method widely used to detect protein and RNA as well as DNA structures in place in the cell, or in
situ, requires expertise
● more rapid assay with higher resolution and flexibility than karyotyping.
● it is limited to the regions complementary to the FISH probes.
● FISH analysis requires a fluorescence microscope that will excite fluorescent emission for the probes
and special filters for detection of fluors emitting at different wavelengths

INTERPHASE FISH
● are used commonly to study prenatal samples, tumors, and hematological malignancies
● fixed cells are permeabilized and exposed to a probe.
● The probe is a 60- to 200-kb fragment of DNA attached covalently to a fluorescent molecule.
● Probes are designed to be complementary to a particular chromosome or chromosomal locus so that
the image under the microscope will correlate with the state of that chromosome or locus.
● dual-color probes, or dual-fusion probes.

Centromeric probes (CEN probes) are designed to hybridize to highly repetitive alpha satellite sequences
surrounding centromeres. These probes detect aneusomy of any chromosome.
Telomeric probes are useful for the detection of chromosome structural abnormalities, such as cryptic
translocations or sub- telomeric deletions that are not easily visualized by standard karyotyping.

SAMPLE PREPARATION FOR INTERPHASE FISH ANALYSIS

1. incubated overnight (aging) after deposition on slides.
2. treated with protease
(to minimize interference from cytoplasmic proteins)
3. fixed with 1% formaldehyde to stabilize the nuclear morphology.
4. dehydrated in graded concentrations of ethanol.

● Paraffin-embedded fixed tissues are dewaxed in xylene before protease and formaldehyde treatment.
● Probes are available commercially
● Both probe and target must be denatured prior to hybrid-ization.
● FISH slides should not be subjected to prolonged light exposure.

METAPHASE FISH
● allows analysis of small regions not visible by regular chromosome banding.
● Probes that cover the entire chromosome, or whole chromosome paints, are
valuable for detecting these small or complex rear- rangements
● spectral karyotyping can distinguish all 23 chromosomes by
chromosome-specific colors.

PREPARATION OF CHROMOSOME FOR METAPHASE FISH

1. The culture of cells for 72 hours.
2. 45 minutes before harvesting, colcemid is added to the cultures to arrest dividing cells in metaphase.
3. Cells are then suspended in a hypotonic medium (0.075 M KCl) and fixed with methanol/acetic acid (3:1).
4. fixed-cell suspension is applied to an inclined slide and allowed to dry.
5. second treatment with 70% acetic acid
6. improve the chromosome spreading and decrease background noise
7. Once the slide is dried, hybridization proceeds
COMPARATIVE GENOME HYBRIDIZATION (CGH)
● Intrachromosomal amplifications or deletions can be detected
● DNA from test and reference samples is labeled and used as a probe on a normal metaphase
chromosome spread
● One advantage of CGH is its capability to identify the location of deletions or amplifications throughout
the genome
● The test DNA is isolated and labeled along with a reference DNA.
● Two colorimetrically distinct cyanine dyes, commonly Cy3 and Cy5, are used as fluorescent labels for
the test and reference
● DNA.
● Cy3 = 550 nm, green
● Cy5 = 650 to 667 nm , red

1. Separate aliquots of test and reference DNA are labeled with different Cy3 and Cy5 dyes,
2. Application to a normal meta- phase spread
3. Test DNA is partially digested with DNase
4. to produce fragments that will bind efficiently to the de- natured DNA in a metaphase chromosome
spread.
5. Although CGH requires advanced technical expertise, it has shown value in identifying recurrent
genomic im- balances not detected by karyotyping.