Cytogenetics

Objectives

• History
• Basic Concepts and Background
• Basic Cytogenetics Laboratory Procedures
• Examining and Analyzing Chromosomes
 What is Cytogenetics

• The study of
chromosome and the
related disease states
caused by abnormal
chromosome number
and or structure.
• Both the number and
structure of the
chromosomes are
analyzed.
 In the beginning,
Cytogenetics did not exist
• Techniques were
pioneered by plant
geneticists
• Progress was slow due to
inability to visualize
human chromosomes as
individual structures
• Each decade since the late
40’s has resulted in
incredible improvements in
the quality of data
available to us for the
analysis and diagnosis of
chromosome abnormalities
Discovering the Chromosomes
• The beginning of human
cytogenetics is generally
attributed to Walther Flemming,
an Austrian cytologist and
professor of anatomy, who
published the first illustrations of
human chromosomes in 1882.
• Flemming also referred to the
stainable portion of the nucleus as
chromatin and first used the term
mitosis. In 1888, Waldeyer
introduced the word
chromosome , from the Greek
words for “colored body,” and
several prominent scientists of the
day began to formulate the idea
that determinants of heredity
were carried on chromosomes.
 Founding Cytogenetics

• After the “rediscovery”

of Mendelian inheritance
in 1900, Walter Sutton
(and, independently at
around the same time,
Theodor Boveri) formally
developed a
“chromosome theory of
inheritance”. Sutton
combined the disciplines
of cytology and genetics
when he referred to the
study of chromosomes as
cytogenetics.
 The 1940’s – The Sex
Revolution
• Dr. Murray Barr and Dr.
Ewart Bertram
discovered sex-
chromatin, now known
as the Barr body, while
working at The
University of Western
Ontario, in 1949.
• A way to clearly
visualize human
chromosomes was yet to
be discovered.
 The 1950’s – Hypotonic
Revolution
• T. C. Hsu discovered the
utility of hypotonic
solution in 1952
• Tijo and Levan reported
the correct chromosome
number in humans in
vitro. This was
confirmed within a year,
in vivo by Ford and
Hamerton (1956).
• They could count now,
but classification was
rough and approximate.
 The 1960’s – The Clinical
Revolution
• Whole Chromosome
syndromes explained
phenotypes.
 The 1970’s – The Banding
Revolution

• Q-banding,
Caspersson, 1970
• G-banding by trypsin
(Seabright, 1971)
• High Resolution
Gbanding, Yunis,
1975
 The 1980’s – The Imaging
Revolution
• Several companies
sprouted at this time,
offering semi-automated
karyotyping and high
quality digital imaging
 The 1990’s – The Color
Revolution
• The return to
fluorescence microscopy
- whole chromosome
painting, SKY, and
unique locus probes
 The 2000’s – The Genomic
Revolution
• BAC and Oligo Arrays
• SNP Arrays
• NextGen Sequencing
 What is a cell?

• Cells are the basic building

blocks of all living things. The
human body is composed of
trillions of cells. They provide
structure for the body, take in
nutrients from food, convert
those nutrients into energy,
and carry out specialized
functions.
• Cells also contain the body’s
hereditary material and can
make copies of themselves.
 What is DNA?
• DNA, or deoxyribonucleic acid, is the
hereditary material in humans and almost
all other organisms. Nearly every cell in a
person’s body has the same DNA. Most DNA
is located in the cell nucleus (where it is
called nuclear DNA).
• The information in DNA is stored as a code
made up of four chemical bases: adenine
(A), guanine (G), cytosine (C), and thymine
(T). Human DNA consists of about 3 billion
bases, and more than 99 percent of those
bases are the same in all people.
• The order, or sequence, of these bases
determines the information available for
building and maintaining an organism,
similar to the way in which letters of the
alphabet appear in a certain order to form
words and sentences.
 What is a gene?

• A gene is the basic physical and functional unit of

heredity. Genes, which are made up of DNA, act
as instructions to make molecules called
proteins.
• In humans, genes vary in size from a few hundred
DNA bases to more than 2 million bases.
• The Human Genome Project has estimated that
humans have between 20,000 and 25,000 genes.
• Every person has two copies of each gene, one
inherited from each parent. Most genes are the
same in all people, but a small number of genes
(less than 1 percent of the total) are slightly
different between people. Alleles are forms of
the same gene with small differences in their
sequence of DNA bases. These small differences
contribute to each person’s unique physical
features.
 What is a chromosome?
• In the nucleus of each cell, the DNA molecule
is packaged into thread-like structures called
chromosomes.
• Each chromosome is made up of DNA tightly
coiled many times around proteins called
histones that support its structure.
• Chromosomes are not visible in the cell’s
nucleus—not even under a microscope—when
the cell is not dividing. However, the DNA that
makes up chromosomes becomes more tightly
packed during cell division and is then visible
under a microscope. Most of what researchers
know about chromosomes was learned by
observing chromosomes during cell division.
• Each chromosome has a constriction point
called the centromere, which divides the
chromosome into two sections, or “arms.” The
short arm of the chromosome is labeled the “p
arm.” The long arm of the chromosome is
labeled the “q arm.” The location of the
centromere on each chromosome gives the
chromosome its characteristic shape, and can
be used to help describe the location of
specific genes.
Chromosome Structure
 How many chromosomes do
people have?
• In humans, each cell
normally contains 23 pairs
of chromosomes, for a total
of 46.
• Twenty-two of these pairs,
called autosomes, look the
same in both males and
females. The 23rd pair, the
sex chromosomes, differ
between males and
females.
• Females have two copies of
the X chromosome, while
males have one X and one Y
chromosome.
 How do cells divide?

• There are two types of

cell division: mitosis and
meiosis. Most of the
time when people refer
to “cell division,” they
mean mitosis, the
process of making new
body cells. Meiosis is the
type of cell division that
creates egg and sperm
cells.
 The Cell Cycle

• The average mammalian

cell cycle lasts about 17–
18h and is the transition of
a cell from one interphase
through cell division and
back to interphase
• The cell cycle is divided
into four major stages. The
first three stages, gap 1
(G1), synthesis (S), and
gap 2 (G2), comprise
interphase. The fourth and
final stage of the cell cycle
is mitosis (M).
•
Mitosis
Mitosis is a fundamental process
for life. During mitosis, a cell
duplicates all of its contents,
including its chromosomes, and
splits to form two identical
daughter cells. Because this
process is so critical, the steps of
mitosis are carefully controlled
by a number of genes. When
mitosis is not regulated correctly,
health problems such as cancer
can result.
 Meiosis
• The other type of cell division,
meiosis, ensures that humans have
the same number of chromosomes
in each generation. It is a two-step
process that reduces the
chromosome number by half—from
46 to 23—to form sperm and egg
cells. When the sperm and egg cells
unite at conception, each
contributes 23 chromosomes so the
resulting embryo will have the usual
46. Meiosis also allows genetic
variation through a process of DNA
shuffling while the cells are
dividing.
PART 2
 What tissues are appropriate
for Chromosome study?
• A tissue that can be
stimulated to undergo cell
division in-vitro
• It is only during mitosis of
the cell cycle that distinct
chromosomes can be
visualized with a light
microscope
• Examples of tissues that
can be studied: chorionic
villi, amniotic fluid,
peripheral blood
(lymphocytes), skin
(fibroblasts), bone marrow
Overview of Cell Culture and
Harvest
Peripheral Blood Culture and
Harvest
 Culture Initiation

• Culture specimen in
suitable culture media
• Blood – RPMI with
mitogen
 Incubation

• Closed system
incubation at 37°C with
increased CO2
environment
• Blood – 72 to 96 hours
 Mitotic arrest

• Arrest cells in the stage

they must be for
standard cytogenetic
analysis: metaphase
• Done by addition of
colchicine to prevent
spindle fiber formation
 Hypotonic treatment

• Treatment with hypotonic

saline solution (KCl) to
increase cell volume so
the chromosomes can fins
space to spread
• Prewarming the hypotonic
solution to 37°C may
increase effectiveness by
speeding up water
transport across cell
membrane possibly
softening the cytoplasmic
membrane
 Pre-fixation

• Addition of 5% Acetic
Acid solution to get rid
of debris (RBC) that
would interfere with
slidemaking and banding
 Fixation

• Carnoys Fixative – 3:1

methanol-acetic acid
• Removes water from
cells, kills and preserves
them, hardening
membranes and
chromatin in
preparation for banding
procedure
 Slide Making

• Once cells have been

fixed, they are dropped
into glass slides and
dried using specific
conditions for optimal
chromosome spreading
and morphology
• Drying chambers are
used to control
temperature, humidity
and air flow conditions
to optimize chromosome
spreading
 Chromosome Staining and
Banding
• Chromosomes are G-
banded to facilitate the
identification of
structural abnormalities.
Slides are dehydrated,
treated with the enzyme
trypsin, and then
stained.
G Banding
Q-banding
Reverse banding
C-banding
AgNOR
 Why do we study
chromosomes?
• To put it simply, chromosomes are important because
they contain the entire (or at least the vast majority of)
genetic information for an organism.
• We search for both the numerical and structural
abnormalities of the chromosomes during analysis.
 Indications For Cytogenetic
Analysis
• Whenever a clinician suspects a patients’
condition/disease is due to a chromosomal abnormality,
he/she should consider a cytogenetic analysis.
 Clinical Indications for
Investigation
of Constitutional Karyotype
Significant family history of:
– chromosome rearrangements
– mental retardation of possible chromosomal origin where
it is not possible to study the affected individual.
 Clinical Indications –
Continued
Patient with:
– primary or secondary amenorrhea or premature menopause
– sperm abnormalities - azoospermia or severe oligospermia
– clinically significant abnormal growth – short stature, excessive growth,
microcephaly, macrocephaly
– ambiguous genitalia
– abnormal clinical phenotype or dysmorphism;
– multiple congenital abnormalities
– mental retardation or developmental delay
– suspected deletion/ microdeletion/ duplication syndrome
– increased risk for a microdeletion syndrome due to a positive family history
– X-linked recessive disorder in a female
– clinical features of a chromosome instability syndrome, including isolated
hematologic findings;
– monitoring after bone marrow transplantation
– a malformed fetus or stillbirth of unknown etiology
– third and subsequent consecutive miscarriage(s) or products of conception from
the fetus.
 Clinical Indications –
Continued
Couple with:
– chromosome abnormality or unusual variant detected at
prenatal diagnosis;
– unbalanced chromosome abnormality in the products of
conception;
– child with a chromosome abnormality or unusual variant;
– infertility of unknown etiology
 Microscopic Analysis

• Chromosomes can be
identified by:
– Their size
– Their shape (the
position of the
centromere). Note:
Chromosomes are
flexible
– Banding patterns
produced by specific
stains (Giemsa)
 Chromosome Structure:
Regions, Bands and Sub-bands
Ideogram
Banding Resolution
Image acquisition and
analysis software
Karyotyping Demo
Normal karyotype?

47,XY,+21
 How and when do the
abnormalities occur?
• Chromosome abnormalities typically
occur as a result of errors in one, or
more, of the following: meiosis, mitosis,
maternal age, or environment.
 Can changes in the number of
chromosomes affect health and
development?
• Human cells normally contain 23 pairs of chromosomes, for
a total of 46 chromosomes in each cell.
• A change in the number of chromosomes can cause
problems with growth, development, and function of the
body’s systems. These changes can occur during the
formation of reproductive cells (eggs and sperm), in early
fetal development, or in any cell after birth.
• A gain or loss of chromosomes from the normal 46 is called
aneuploidy.
 Can changes in the structure of
chromosomes affect
health and development?
• Changes that affect the structure of chromosomes can cause
problems with growth, development, and function of the body’s
systems. These changes can affect many genes along the
chromosome and disrupt the proteins made from those genes.
• Structural changes can occur during the formation of egg or sperm
cells, in early fetal development, or in any cell after birth. Pieces
of DNA can be rearranged within one chromosome or transferred
between two or more chromosomes.
• The effects of structural changes depend on their size and
location, and whether any genetic material is gained or lost. Some
changes cause medical problems, while others may have no effect
on a person’s health.
Chromosomal Mosaicism
 Acquired Abnormalities

• Many cancer cells also have changes in their

chromosome structure. These changes are not
inherited; they occur in somatic cells (cells other than
eggs or sperm) during the formation or progression of a
cancerous tumor.
 The role of Cytogenetics

• Can help clinicians with reaching a diagnosis, as an ECG

is to a cardiologist or an EEG is to a neurologist, or an X-
ray is to a pulmonologist
• Diagnostic findings can affect counselling for families –
e.g. Type of translocation in Down syndrome patients
FISH principle
Thank You for
Listening!