Topic 4: Cytogenetics

Cytogenetics
i. Understand the role of cytogenetics in modern
genetics
ii. Describe the structure of a typical human
chromosomes
iii. Be able to analyze/interpret various karyotypes
iv. Explain how cells can be obtained and treated for
chromosome analysis
v. Understand how variations in chromosome
number can impact development, providing
examples
vi. Describe various structural abnormalities that
can occur within chromosomes
vii. Explain the FISH technique and how/why it is
applied to cytogenetics
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Topic 4: Cytogenetics

What is Cytogenetics
“The study of the structure and function of
chromosomes, and their role in genetics”

• 1882: Walther Flemming started the field of

human cytogenetics by describing chromatin
as the rod-shaped bodies that he saw in the
nucleus of cells.

Chromosomes released from

a lysed cell. Treating the
chromosomes with a dye
called Giemsa causes the
chromosomes to appear
dark purple (“G-banded”).
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Topic 4: Cytogenetics

Chromosomal Comparisons
• Chromosome analysis is a
powerful and useful technique
in human genetics
• The number of chromosomes
in the nucleus of an organism
is characteristic for a species
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Topic 4: Cytogenetics
* protein-coding genes

Size Doesn’t Matter…

• Chromosome number vs. genome size – not directly comparable
• Homo sapiens: 23 chromosomes, ~3.2 billion bp (~20,000 genes*)
• Canis familiaris: 39 chromosomes, ~2.4 billion bp (~25,000 genes*)
• Paris japonica: 40 chromosomes, 149 billion bp (? genes)
• Sorangium cellulosum: 1 chromosome, 13 million bp (~9,000 genes*)
• Synthetic bacteria: 1 chromosome, 531,000bp (437 genes*)
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Topic 4: Cytogenetics

Why do we Care about Chromosomes?

Consider the following:
An imbalance (i.e., too many or too few) of even a small number of genes is known to
have profound effects on normal development.

Therefore, since chromosomes carry the

genes, it might be expected that any kind of
chromosome abnormality is problematic.
Indeed, with rare exceptions this is the case.

Chromosome abnormalities are the leading

known cause of pregnancy loss and
congenital birth defects in our species
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Topic 4: Cytogenetics

Why do we Care about Chromosomes?

Examples of common aneuploidy disorders:
• Down Syndrome (Trisomy 21)
• Edward’s Syndrome (Trisomy 18)
• Klinefelter Syndrome (XXY)
• Turner Syndrome (X)
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Topic 4: Cytogenetics

Review: DNA Organisation

• DNA is wrapped 1.67 times around a group of special proteins called histones
• DNA in contact with the histones is called core DNA, whereas the DNA between two
nucleosomes is called linker DNA
• The distance between nucleosomes can change, leading to more compact chromatin
(heterochomatin) or more loosely packed chromatin (euchromatin)
• There are 8 histone units in each nucleosome: 2 of each H2A, H2B, H3 and H4

Core DNA

Histone octamer
Linker DNA
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Topic 4: Cytogenetics

Review: DNA Organisation

• Histones: proteins that act as
‘spools’ for DNA
• Chromatin: coiled &
compacted mass of DNA
• Euchromatin
• Loosely coiled (DNA is more
accessible)
• DNA can be transcribed –
genes are expressed
• Heterochromatin
• Tightly packed (DNA is
inaccessible)
• DNA cannot be transcribed
– genes are not expressed
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Topic 4: Cytogenetics

Review: DNA Organisation

• Histones: proteins that act as
‘spools’ for DNA
• Chromatin: coiled &
compacted mass of DNA
• Euchromatin
• Loosely coiled (DNA is more
accessible)
• DNA can be transcribed –
genes are expressed
• Heterochromatin
• Tightly packed (DNA is
inaccessible)
• DNA cannot be transcribed
– genes are not expressed
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Topic 4: Cytogenetics

Chromosome Structure
• # of autosomes in a gamete = ______ in a somatic cell = ______

• # of sex chromosomes in a gamete = ______ in a somatic cell = ______

• Total # of chromosomes in a gamete = ______

• Total # of chromosomes in a somatic cell = ______

• # of chromosomes in a trisomic cell = ______

• # of chromosomes in a monosomic cell = _______

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Topic 4: Cytogenetics

Chromosome Structure

Chromatin fiber consists of DNA and proteins (i.e., histones):

• heterochromatin = tightly packed; transcriptionally inactive
• euchromatin = loosely packed; transcriptionally active

Centromere visible as a constricted region

Chromatid

The two, identical sister

chromatids are clearly visible.
Chromosome
Electron micrograph of human chromosome 12
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Topic 4: Cytogenetics

Chromosome Structure

Short arm: “p” (petit) p

Telomeres:
Centromere: 6 bp sequence
Highly repetitive (TTAGGG) repeated
“ 1000s of times
satellite DNA”
(171 bp repeats)
Long arm: “q” q
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Topic 4: Cytogenetics

Chromosome Structure: Centromeres

• Centromere
• A region of a chromosome…
i. joining sister chromatids, and
ii. to which microtubule fibers attach during cell division

• The location of a centromere gives a chromosome its characteristic

shape

• Centromeres contain tandem repetitive sequences, called

satellite DNA
i. These repetitive sequences bind special proteins called “cen-
proteins”
ii. Cen-proteins are essential for the attachment of spindle fibers
during cell division Note the centromere position of
the chromosomes pictured above.
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Topic 4: Cytogenetics

Chromosome Structure: Centromere Position

3 Arrangements:

Metacentric
• A chromosome that has a centrally placed
centromere

Submetacentric
• A chromosome whose centromere is placed
closer to one end than the other

Acrocentric
• A chromosome whose centromere is placed
very close to, but not at, one end Chromosome 3 Chromosome 17 Chromosome 21
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Topic 4: Cytogenetics

Chromosome Structure: Telomeres

A telomere is a repeating sequence of
double-stranded DNA located at the ends of
chromosomes.

• Greater telomere length is associated with

immortalized cell lines such as embryonic stem
cells & cancer cells.

• As cells divide and differentiate throughout the

lifespan of an organism or cell line, the
telomeres become progressively shortened.
Human chromosomes treated
to highlight the telomeres
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Topic 4: Cytogenetics

Types of Chromosomes
2 kinds of chromosomes:

Autosomes
• Chromosomes other than the sex chromosomes
• In humans, chromosomes 1 to 22 are autosomes
• Even the smallest autosome carries >200 genes

Sex Chromosomes
• In humans, the X and Y chromosomes that are involved in sex determination
• The X chromosome is composed of ~150 million bp and has ~1000 genes.
• The Y chromosome is composed of ~55 million bp and has ~60 genes.
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Topic 4: Cytogenetics

Human Karyotype
• Karyotype: the complete set of chromosomes from
a cell that has been photographed during cell
division & arranged in a standard sequence

• Chromosomes are arranged according to size &

centromere position

• The bands you see along each chromosome are the

result of staining the chromosomes with Giemsa
stain.
• We say the chromosomes are “G banded”
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Topic 4: Cytogenetics

Human Karyotype
Band
6
• Chromosomes are artificially Region
5
4
stained in the laboratory. 3 3
2
1
• Each chromosome displays a 2 2
1 2 3 4 5 6 1
distinctive banding pattern. Arm
3
p 1 12
q 1
• Allows any region to be 1 2

identified by a descriptive 7 8 9 10 11 12 12 locus 1q2.4

3
2
address (chromosome number, 4
5
arm, region, and band) 1
• e.g. arrow is pointing to the locus 13 14 15 16 17 18 3 2
1q2.4 12
4
43
19 20 21 22 Y 1
X
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Topic 4: Cytogenetics

Human Karyotype
Any nucleus can be used to make
karyotype
• Lymphocytes, skin cells, cells from biopsies,
tumor cells
Sampling cells before birth
• Amniocentesis
• Chorionic villus sampling (CVS)
• Fetal cells from mother’s blood
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Topic 4: Cytogenetics

Human Karyotype
Note: Red blood cells do NOT have a
nucleus, therefore white blood cells
are needed for analysis
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Topic 4: Cytogenetics

Information Obtained from Karyotypes

• Number of chromosomes
• Sex chromosome content
• Presence or absence of individual
chromosomes
• 1959: Discovery that Down syndrome is caused
by an extra copy of chromosome 21
• Nature & extent of large structural
abnormalities
Question:
What is abnormal about
this karyotype?
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Topic 4: Cytogenetics

Human Karyotype - Amniocentesis

• A method of sampling the fluid Removal of about
20 ml of amniotic
fluid containing
surrounding the developing fetus by suspended cells
that were sloughed
inserting a hollow needle and withdrawing off from the fetus

suspended fetal cells and fluid

A few biochemical
• Used in diagnosing fetal genetic and analyses with some Centrifugation
of the amniotic fluid
developmental disorders
• Usually performed in the 16th week of Quick determination of
fetal sex and analysis
Fetal cells
pregnancy of purified DNA

Biochemical analysis for Growth for

the presence of alleles that several days
cause many different in culture
metabolic disorders medium

Karyotype analysis
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Topic 4: Cytogenetics

Human Karyotype - CVS

• Chorionic Villus Sampling is a
method of sampling fetal chorionic
cells by inserting a catheter through
the vagina or abdominal wall into
the uterus
• Used in diagnosing biochemical and
cytogenetic defects in the embryo
• Usually performed in the eighth or ninth
week of pregnancy
• BUT: risk of miscarriage is higher than
with an Amnio
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Topic 4: Cytogenetics

Human Karyotype – Fetal Cells from Mother’s

Blood
• It is now possible to
isolate fetal cells that
pass into the mother’s
bloodstream (placental
cells, white blood cells,
immature red blood
cells) for genetic testing,
rather than using more
invasive techniques
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Topic 4: Cytogenetics

Human Karyotype
• We can now provide pre-natal
screening for various
chromosomal abnormalities
(aneuploidies and
polyploidies)
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Topic 4: Cytogenetics

Chromosomal Abnormalities
Two major types of chromosomal changes

1. Change in chromosomal number

• Polyploidy: A chromosomal number that is a
multiple of the normal haploid chromosomal set
• Aneuploidy: A chromosomal number that is not
an exact multiple of the haploid set

2. Change in chromosomal arrangement /

structure
• Deletions, translocations, duplications, etc.
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Topic 4: Cytogenetics

Chromosomal Abnormalities
Polyploidy and aneuploidy are major causes of
reproductive failure in humans
• Polyploidy is seen only rarely in live births (usually X or Y)

• Rate of aneuploidy in humans is much higher than in other

primates and mammals (reasons for the difference are
unknown)
• Aneuploidy is the leading cause of reproductive failure in
humans
• Results in spontaneous abortions and birth defects

• Aneuploidy also is associated with most cancers Question: how does polyploidy happen?
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Topic 4: Cytogenetics

Polyploidy

Triploidy
Triploidy
• Chromosomal number that is three times
the haploid number, having three copies of
all autosomes and three sex chromosomes
• Can arise from either gametogenesis

Tetraploidy
• Chromosomal number that is four times the

Tetraploidy
haploid number, having four copies of all
autosomes and four sex chromosomes
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Topic 4: Cytogenetics

Causes of Aneuploidy
Note: monosomy & trisomy involve the loss / gain
Caused by non-disjunction of a single chromosome to a diploid genome

• Failure of homologous chromosomes

to separate properly during meiosis
Monosomy
• A condition in which one member of a
chromosomal pair is missing; one less
than the diploid number (2n – 1)

Trisomy
• A condition in which one chromosome
is present in three copies, and all
others are diploid; one more than the
diploid number (2n + 1)
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Topic 4: Cytogenetics

Effects of Monosomy & Trisomy

Autosomal monosomy is a lethal
condition
• Eliminated early in development (spontaneous
abortion)

Autosomal trisomy is relatively common

• Most result in spontaneous abortion
• Three types can result in live births (13, 18, 21)
• Patau Syndrome, trisomy 13 (47,+13)
• Edwards Syndrome, trisomy 18 (47,+18)
Patau Syndrome (left) and Edwards
• Down Syndrome, trisomy 21 (47, +21) Syndrome (right) are both lethal conditions
associated with numerous physiological and
neurological abnormalities
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Topic 4: Cytogenetics

Risks for Autosomal Trisomy

Autosomal trisomy is selected against less
stringently than autosomal monosomy
• Cases of partial development & live births of
trisomic individuals occur
• Only individuals with trisomy 21 survive into
adulthood
Maternal age is the leading risk factor for
trisomy
• Trisomy 21: 94% of non-disjunctions occur in the
mother, 6% in the father
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Topic 4: Cytogenetics

Risks for Autosomal Trisomy

Why is maternal age such a risk factor?
• Meiosis is not completed until ovulation
• Intracellular events may increase risk of
non-disjunction, resulting in aneuploidy
• Maternal selection
• Embryo-uterine interactions that normally
abort abnormal embryos become less
effective

Topic 4: Cytogenetics
Aneuploidy of Sex Chromosomes
• Aneuploidy of sex chromosomes
involves both X & Y chromosomes
• A balance is needed for normal
development
• At least one copy of the X chromosome is
required for development
• Increasing numbers of X or Y chromosomes
causes progressively greater disturbances in
phenotype and behavior
33
Male
• Klinefelter (47,XXY): 1/1000 males
• 47,XYY: 1/1000 males
• 46,XX males: 1/20,000 males
Female
• Turner (45,X): 1/5000 females
• Trisomy X (47,XXX): 1/1000 females
• 46,XY females: 1/20,000 females
• Androgen insensitivity (testicular
feminization): 1/20,000 females
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Topic 4: Cytogenetics

Aneuploidy of Sex Chromosomes

Klinefelter Syndrome
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Topic 4: Cytogenetics

Aneuploidy of Sex Chromosomes

Turner Syndrome
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Topic 4: Cytogenetics

Chromosome Structural Abnormalities

Chromosomes can lose, gain, or rearrange
segments!
Changes in the arrangement of
chromosomes:
i. Deletions
ii. Duplications
iii. Inversions
iv. Translocations

NOTE: mainly caused by mistakes of

crossing-over during meiosis!
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Topic 4: Cytogenetics

Structural Abnormalities: Deletions

Deletions
• Involve a loss of chromosomal material
• Examples:
• Cri du chat syndrome
• DiGeorge Syndrome
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Topic 4: Cytogenetics

Structural Abnormalities: Deletions

Cri du chat syndrome
• Deletion of the short arm (p) of chromosome 5
• Associated with an array of congenital malformations
• Most characteristic: infant cry that resembles a meowing cat
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Topic 4: Cytogenetics

Structural Abnormalities: Deletions

DiGeorge syndrome
• a.k.a. 22q11.2 deletion syndrome
• Deletion in long arm (q) of chromosome 22
• Suspected upon looking at karyotype. Can
be confirmed by FISH.
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Topic 4: Cytogenetics

FISH
Fluorescent In Situ Hybridization
• Probes are designed to be complimentary to a target sequence (e.g. chromosomal locus
associated with disease)
• Probe is labeled with a fluorescent tag that allows us to see where the probe is binding.

FISH used to detect and diagnose trisomies (e.g., Down syndrome)

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Topic 4: Cytogenetics

FISH
Fluorescent In Situ Hybridization
• Probes are designed to be
complimentary to a target sequence
(e.g. chromosomal locus associated
with disease)
• Probe is labeled with a fluorescent
tag that allows us to see where the
probe is binding.
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Topic 4: Cytogenetics

Structural Abnormalities: Duplications

Duplication
• When any part of the genetic
material is present more than
once in the genome
• May be a single locus (e.g. one
gene), or a large piece of a
chromosome
• May result in gene redundancy
• Important source of genetic
variability during evolution
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Topic 4: Cytogenetics

Structural Abnormalities: Duplications

Duplication
• Due to crossing over problems
(unequal crossing)
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Topic 4: Cytogenetics

Structural Abnormalities: Inversions

Inversions
• Occur when a segment of DNA
is turned around 180 degrees
within a chromosome
• When there are two breaks in a
chromosome, then rejoined (get
inversion loop)
• No loss of genetic material
• May involve the centromere
(pericentric inversion), or not
(paracentric inversion)
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Topic 4: Cytogenetics

Structural Abnormalities: Translocations

Translocations
• Involve exchange of chromosome parts
• Often produce no overt phenotypic
effects
• Can result in genetically-imbalanced &
aneuploid gametes
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Topic 4: Cytogenetics

Structural Abnormalities: Translocations

Translocations
• Balance of genes is still normal (nothing has been
gained or lost)
• Can alter phenotype as it places genes in a new
environment
• Can cause difficulties in egg or sperm development
and normal development of a zygote

9-22 translocation,
which is associated
with leukemia
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Topic 4: Cytogenetics

Structural Abnormalities: Translocations

Robertsonian Translocation
• When an acrocentric chromosome’s q-arm translocates onto another acrocentric
chromosome to make one very large chromosome & one very small one
• The small one may actually be removed if it contains non-essential genes
• You can keep all your DNA and have only 44 chromosomes!
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Topic 4: Cytogenetics

Structural Abnormalities: Translocations

Robertsonian Translocation
• e.g. Translocation of chromosomes
13 & 14
• End-to-end fusion of the two
chromosomes
• Shown: balanced rearrangement
(no net gain or loss of genetic
material) -> normal phenotype.
• However: risk for an abnormal
child or spontaneous pregnancy
loss is increased
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Topic 4: Cytogenetics

Review
i. How and why is cytogenetics applied to fertility clinics (i.e., in association with IVF)?
ii. What are three component structures of a chromosome and what is the function of each?
iii. Compare and contrast the structure of chromosomes in interphase vs the M phase.
iv. Describe the structure and function of histones.
v. Explain how cells are obtained from children and adults vs. from a fetus for chromosome analysis
vi. Why does oogenesis have a higher risk of transmitting aneuploidies than spermatogenesis?
vii. Compare and contrast reciprocal vs Robertsonian translocations.
viii. Describe the cytogenetics of cri-du-chat syndrome.
ix. Explain the FISH technique and how/why it is applied to cytogenetics.