BASIC CYTOGENETICS

TRI INDAH WINARNI, MD, PhD

CENTER FOR BIOMEDICAL RESEARCH (CEBIOR) FACULTY of MEDICINE
DIPONEGORO UNIVERSITY

Presented at Medical Genetics Workshop. Semarang,

November 29th, 2016
 What is chromosome?

Chromosome visualization

Chromosomal abnormalities

Chromosomal Rearrangement & Cancer

Cytogenetic
CHROMOSOMES
The most important macromolecule in the cells
which carry genetic information (genes)
The DNA sequence for a single trait is called a
gene, a sequence of DNA bases that specify the
order of amino acids in a protein
Genes are responsible for hereditary
characteristics (diverse among us)
Each chromosome contains a few thousand
genes 20.000 gene in human chromosome
has been found
 Chroma: colour, soma: body, coloured body that visualized by light
microscope

In 1956, 46 chromosome in human cells was identified with 22

autosomal pairs and 2 sex chromosomes - 2 X chromosomes for
females or an X and a Y chromosome for males

During interphase they are not visible, during metaphase stage it

highly condensed and visible under the light microscope
Chromatin
DNA & protein (histone) complex that
makes up chromosomes
Two types of chromatin that can be distinguished:
Euchromatin, which consists of DNA that is actively
expressed as protein.
Heterochromatin, which consists of mostly inactive
DNA. Heterochromatin can be further distinguished
into two types:
1. Constitutive heterochromatin, which is never expressed.
It is located around the centromere and usually contains
repetitive sequences
2. Facultative heterochromatin, which is sometimes
expressed.
Chromatid
A single DNA double helix that packed into a chromosome

During cell division consist of two identical genetic material, called

sister chromatids joined at centromere

Centromere is responsible very important for the movement of

chromosomes during cell division

Each chromosome has 2 arm: long arm (q arm) and short arm (p
arm)
Chromosome Terminology
 What is Chromosome?

Chromosome visualization

Chromosomal abnormalities

Chromosomal Rearrangement & Cancer

Cytogenetic
CULTURE
5-10 ml blood collected in heparin tube care must be taken
to avoid extremes temperature (stored at room or refrigerated >
4C)

Heparin red cells in the bottom, while the white cells and
platelets forming thin layer on the top (buffy coat)

5-6 drops with a sterile pasteur pipette added to the media

 Medium (MEM, TC 199, RPMI) added with at least:
1. FCS/FBS promote cells growth
2. PHA mitotic stimulant
3. Antibiotic to prevent contamination
Incubate culture for 72 hours in CO2 cell culture incubator

Add Colcemid (about 45 min before harvesting)

arresting the cells in metaphase stage
HARVEST
3 Steps of harvesting procedures:
1. Stopping the cells division in metaphase stage by adding
colcemid, prevents the cells from entering anaphase stage
2. Exposing the cells to a hypotonic solution (KCL) uptake
the water through semi-permeable cell membrane liquid-
filled balloon
3. Fixative (Carnoys/Methanol + Acetic acid) removes water
from the cells & enhance chromosome morphology and
their ability to take up the stain
4. Repeat step 2 & 3 three times to make the chromosome
ready to be spread
SLIDE PREPARATION
The most critical steps in visualizing a good metaphase
spread cells
The cells membrane should keep remain intact, if not the
chromosome may spill out causing chromosome loss
Next step is making slide old enough in order get better
stained (manipulative or natural way)
G-banding (Giemsa) to see the bands (dark and
light/hetero and euchromatin)
Technique to visualize chromosome
1. GTG/G banding: (trypsin induced Giemsa stain):
differentiates light and dark bands
2. C-banding: (centromere stain) to demonstrate constitutive
heterochromatin & centromere region darkly stained
3. Silver-banding/Ag-NOR banding: stain short arms of all
acrocentric chromosomes (satellites), except Y
chromosome
Seven groups of chromosomes based on size and
centromere location
Group A: chromosome 1-3, large, metacentric
Group B: chromosome 4-5, large, submetacentric
Group C: chromosome 6-12 and X, medium, submetacentric
Group D: chromosome 13-15, medium, acrocentric
Group E: chromosome 16-18, short, submetacentric
Group F: chromosome 19-20, short, metacentric
Group G: chromosome 21-22 and Y, short, acrocentric
Idiogram of G-banded chromosome
Chromosome spread with chromosomes shown by bright field G-banding

X
Crossing over

X
Karyotype shown by bright field
G-banding of chromosomes
Karyotype shown by bright field
G-banding of chromosomes
How to analyze the aberration
 What is Chromosome?

Chromosome visualization

Chromosomal abnormalities

Chromosomal Rearrangement & Cancer

Cytogenetic
CHROMOSOMAL ABNORMALITY
1. STRUCTURAL ABNORMALITY caused by crossing
over with error reunion in Meiosis I
Translocation, inversion, deletion, duplication

2. NUMERICAL ABNORMALITY caused by non-

disjunction in Meiosis I & II
Trisomy, Klinefelter & Turner syndrome
MEIOSIS
 2N,2C 2N,4C

1N,2C
Homolog chromosome pull apart
 1N,2C

Sister chromatids pull apart

1N,1C

There is transient interphase/NO chromosome replication

STRUCTURAL ABNORMALITY
BALANCED/Carrier
UNBALANCED gain OR loss
NUMERICAL ABNORMALITY
UNEUPLOIDY (MONO/TRISOMY)
1. Meiotic non-disjunction in the germ cells
2. Mitotic Error will cause mosaicism
In primitive germ cells: germ line mosaicism
During early post-zygotic period: somatic mosaicism
Mechanisms:
Spindle assembly and disassembly checkpoint (SAC) error due to
kinetochore dysfunction
Multipolar (mitosis/meiosis) spindle formation
Error in separation OR Meiotic non
disjunction aneuploidy
(monosomy/trisomy)

non-viable fetus/miscarriage /fetal

death/stillbirth/
multiple anomaly
MOSAICISM
SOMATIC MOSAICISM
Presenting two or more cell lines which derived from a
single zygote/the same genetic origin.
Example: 46,XX/47,XX,+21 (80%)

Symptomatic = clinical implication, depend on the

percentage of the abnormality

Error in mitotic separation OR Mitotic non-disjunction

Time: post-zygotic period

MITOTIC ERROR MOSAIC

Mosaic: 46/47
How to write the results (ISCN)
Normal Karyotype
46,XY
46,XX
Aneuploidy
47,XXY
45,X
47,XX, +21
Mosaicism
47,XX,+21 (60%)/46,XX (40%)
45,X (30%)/46,XX (70%)
How to write the results
Chromosomal aberration/derivation
Translocation
46,XY,t(2;16)(p22;q23) (balanced)
45,XX,der(13;21)(q10;q10) (robertsonian)
46,XX,der(5)t(5;12)(p14;q23) (unbalanced)
Isochromosome
46,X,i(Xq)
Deletion
46,XY,del(13)(q31.1q33.1)
 What is Chromosome?

Chromosome visualization

Chromosomal abnormalities

Chromosomal Rearrangement & Cancer

Cytogenetic
Chromosomal Abnormalities in Human Cancer

1. Balanced chromosomal rearrangements leads to chimeric fusion gene and chimeric

protein expression with new OR altered activity
2. Chromosomal imbalances
a) genomic gain: complete or partial trisomies & intrachromosomal or
extrachromosomal amplifications (homogeneously staining regions (HSR) and double-
minute chromosome (dmin).
b) genomic losses: genomic loss include monosomy and deletion
CHROMOSOMAL REARRANGEMENT in CANCER CYTOGENETIC
To date, 500 tumor-specific translocations have been identified in
tumor cells
There is a higher incidence of cancer deaths due to solid tumors in 80%
and in 10% of hematological malignancies
1. Simplex & Balanced Rearrangement
Formed a fusion proteins that activate oncogene and disruption
of tumor supressor gene
Common in hematologic malignancy
Fist described in 1960: Ph chromosome (t(9;22)) BCR-ABL
activate oncogene (tyrosine kinase) found in various leukemia
SIMPLE BALANCE REARRANGEMENT IN CANCER
CHROMOSOMAL REARRANGEMENT in CANCER CYTOGENETIC

2. Complex Rearrangement
Commonly found in solid tumor
Chromoanagenesis, a mitotic segregation error which cause
ten-hundreds of fusion mapping in one or few chromosomal
loci that lead genomic gains (trisomy, HSR, double minute)
and losses (monosomy, deletion)
In a single catastrophic event
Mechanism for the creation of complex chromosomal rearrangements by non-
homologous end joining (NHEJ). Chromothripsis (chromosome shattering) leads to the
simultaneous creation of many double strand breaks, shattered fragments are stitched back
together though NHEJ to chromoanagenesis. Broken DNA fragments may joined together
to form circular, extra-chromosomal double minute chromosomes that often harbor
oncogenes/amplification, resulting in a dramatically increased copy number of DNA
fragments on these chromosomes.
TECHNIQUES for CHROMOSOMAL REARRANGEMENTS IDENTIFICATION
Spektral karyotype analysis of solid cancer. Metafase and karyotype show high level of
aneuploidy and complex rearrangement. Abnormal chromosomes are identified by the presence
of more than one color, while, unique color pattern show various chromosome involve in
rearrangement.
 Evolution of Cytogenetic Methods for the Study of Chromosome
Aberration in Cancer
A. G-band karyotype B. Spectral Karyotyping C. Chromosome based
CGH D. Oligo array CGH found microdeletion
The more advance the technique the more better capture the
cause