Ecole des Hautes Etudes
de Biotechnologie et de Santé - Casablanca
Cytogenetics and chromosomal disorders
Module de la génétique humaine et analyse génomique et protéomique
Master biotechnologie médicale et bioinformatique: S3
Année universitaire : 2022-2023
Pr.Lamiae El khattabi
OBJECTIFS
0. Knowledge Assessment
1. Recall: Cell Cycle – Chromosome-chromatine Structure -
2. Conventional Cytogenetics: Karyotype
3. Molecular Cytogenetics: FISH, CGH
4. Chromosomal Aberrations
5. Prenatal Diagnosis
6. Onco-hematological Cytogenetics
KNOWLEDGE ASSESSMENT
Q1) Explain in a few lines the principle of PCR (Polymerase Chain Reaction).
Q2) What are the main steps in the synthesis of a protein from a eukaryotic gene?
Q3) Can we study the mRNA of a given gene from any tissue?
Q4) Is the entire sequence of a human gene coding? Provide some keywords on this topic.
Q5) Can a chromosomal deletion of 1 kilobase be observed on a standard karyotype? Justify.
Q6) Can a single fragment of DNA of 200 base pairs be sequenced? Justify. (8
Q7) How are DNA fragments of different sizes separated? Explain.
Q8) Where is DNA located in the human cell?
Q9) Are all RNAs translated into proteins? (Justify your answer.)
Q10) Can you mention an example of a genetic disease: the name of the disease, mode of
transmission, and the name of the gene if known?
Cell Cycle
CHROMOSOME-CHROMATINE STRUCTURE
"Chromatine" and "chromosomes" are two
different aspects of the same entity...
"Loosely condensed and lightly
colored structure, known as
'open': euchromatin (EC)“
"Tightly condensed and strongly
colored structure, known as
'closed': heterochromatin (HC)"
A "chromosome band" refers to any identifiable chromosomal structure.
G-bands and R-bands have specific properties.
G-bands and R-bands are characterized by different-sized loop compaction, reflecting the
specificities of the DNA they contain.
On appelle « bande chromosomique » toute structure chromosomique identifiable.
Les bandes G et R ont des propriétés spécifiques.
Les bandes G et R se caractérisent par une compaction en boucles de tailles différentes,
reflétant les spécificités de l'ADN qu'elles contiennent.
Human Genome
~52% repeated DNA
Unique sequences
1
2
Definition:
The eukaryotic chromosome is a long molecule of DNA
and associated proteins that carries only a portion of
the genetic information.
The centromere
Definition: A chromosome's constriction site whose functions are essential
to the life of eukaryotic cells, especially (1) connecting the two sister
chromatids between replication and anaphase, (2) ensuring protein assembly
of the kinetochore at its level, which is responsible for (3) the equitable
segregation of sister chromatids between the two daughter cells during cell
division. The centromeres of most eukaryotic organisms consist of repeated
DNA sequences and bear specific epigenetic marks.
Steps in the creation of a karyotype
Microscopic observation
Classification of chromosomes in a karyotype
Classification of chromosomes in a karyotype
Metaphase plate Sorted according to:
• Size
• Centromere position
• Band staining
Classification of chromosomes in a karyotype
According to the Size
From largest to smallest
Chromosome length (l)
Size :
the sum of all the chromosomes in the set
Classification of chromosomes in a karyotype
According to the centromere position
T: Télophase M: Métaphase
Defined by
The centromeric index = p/(p+q)
Metacentric; CI=0.5
Submetacentric; CI=0.3
Acrocentric; CI=0 (13, 14, 15, 21, 22)
Classification of chromosomes in a karyotype
➢ Based on the two criteria of size
and centromere position, there
are 7 groups of chromosomes: A -
G.
➢ However, chromosomes within
the same group cannot be
distinguished.
Classification of chromosomes in a karyotype
Based on band staining
The GIEMSA stain produces a uniform pink-purple coloration (S)
Adoption of specific marking or banding techniques
(denaturation)
S: without denaturation
Appearance of a series of light and dark bands along the
G: denaturation with trypsin
chromosome.
R: denaturation with heat
Both techniques are
complementary and yield
reciprocal marking, with a
dark band in one being light
in the other
Classification of chromosomes in a karyotype
Based on band staining
C: Staining with
Barium Sulfate
highlights the
centromeric regions.
The banding pattern is
characteristic of each
chromosome
("barcode").
Chromosome Nomenclature
The bands are cataloged according to an international
nomenclature "ISCN":
7p14
• Each arm is divided into 1 to 4 regions, each region
into bands, and each band into sub-bands.
• The numbering starts from the centromere.
• Writing the position of a band on a chromosome:
Chromosome, arm, region, band.
7q32 Band 2 of region 3 of the long arm (q) of chromosome 7
« ISCN »: international system for human cytogenetic nomenclature
Chromosomal Formula of a Karyotype
The chromosomal formula is the way to express the result of the karyotype and is
deciphered as follows:
"Number of chromosomes per cell", "List of present sex chromosomes", ± "List of
anomalies found”
Example:
Trisomy 21: 47,XY,+21
47 chromosomes per cell, including
one X chromosome and one Y
chromosome, + an extra
chromosome 21.
NORMAL HUMAIN KARYOTYPE
Male karyotype in R banding 46,XY Female karyotype in R banding 46,XX
Limitations of Conventional Cytogenetics
- obtention de métaphases
- résolution : 1 bande
•Benefits of Molecular Cytogenetics...
3. MOLECULAR CYTOGENETIC:
FISH = FLUORESCENCE IN SITU HYBRIDIZATION
HYBRIDATION IN SITU EN FLUORESCENCE
➢Diagnostic tool to specifically characterize chromosomal micro-rearrangements <
3 Mbps, undetectable by karyotyping.
➢Based on the use of the specificity of base-to-base pairing of the DNA molecule
to identify a fragment or the entirety of a chromosome.
➢Hybridization of labeled specific sequences (probes) on an entire genome:
centromeres, sub-telomeric regions, and specific loci.
➢Detection of deletions, identification of translocations, or other chromosomal
rearrangements.
FISH : Differents types of probes
FISH : Differents types of probes
FISH : Caryotype spectral ou SKY ou multi-Fish :
➢ it allows for the visualization of all
23 pairs of human chromosomes at
once. Each pair of chromosomes is
labeled with a different fluorescent
color, enabling researchers or
clinicians to easily identify and
distinguish between each
chromosome pair. This is
particularly useful for identifying
structural chromosomal
abnormalities, such as
translocations, deletions, or
duplications.
3. MOLECULAR CYTOGENTIC:
CGH = HYBRIDATION GÉNOMIC COMPARATIVE
PRINCIPE :
➢ Comparison of the number of molecules from the same amount of DNA from two individuals
by labeling each with a different fluorochrome and then co-hybridizing them onto metaphase
chromosomes of a control or onto a microchip made up of DNA sequences.
➢ After the hybridization step, the signals generated by the two fluorochromes are digitized, and
a report of their respective intensity is established at the level of the bands of each of the
chromosomal pairs.
➢ The intensity ratio of the two fluorochromes at the level of a band will have a theoretical value
close to 1 if the number of molecules from the two genomes is identical at the level of the
observed band.
➢ In the case of a deletion, this theoretical ratio will be 0.5.
➢ In the case of trisomy, the ratio is 1.5.
CGH sur métaphase CGH sur micropuce
QUATRE ÉTAPES:
7
4. PRINCIPALES ANOMALIES CHROMOSOMIQUES:
The genome is very stable but can
undergo variations:
• Quantitative (number of
chromosomes)
• Qualitative (structure of the
chromosomes)
4. MAIN CHROMOSOMAL ANOMALIES:
A chromosomal anomaly refers to any rearrangement in the number or structure of
the chromosomes. Chromosomal anomalies can be:
• Constitutional: chromosomal anomalies that occurred before fertilization.
• Acquired: chromosomal anomalies that appeared during one's lifetime.
• Autosomes and/or gonosomes.
• Homogeneous: all cells show the chromosomal anomaly.
• Mosaic: a fraction of the cells is abnormal.
• Balanced: neither loss nor gain of genetic material → no consequences on the
phenotype.
• Unbalanced: clinical manifestations vary depending on the degree of gain or
loss.
Trisomy 21 : Trisomy 13: 47,XX,+13
47,XY,+21
Klinfelter: 47,XXY Turner: 45,X0
Number Anomalies
Result from poor chromosome segregation during
cell division:
• mitosis
• or one of the divisions of meiosis
No disjonction (ND) in the first division
1st Case 2nd Case
Premature separation
of sister chromatids
Gamete disomic nullisomic disomic normal normal nullosomic
Zygote trisomiy monosomic trisomy normal normal monosomy
During
fertilization
Disomic ovum
nullisomic ovum
The occurrence of autosomal trisomy
results in nonviable zygotes except for
trisomies 21, 13, 18, and 8 (+/- viable).
The only viable monosomy is
Turner Syndrome.
Zygote trisomic Zygote monosomic
Non-disjunction during the first meiotic division of spermatogenesis.
➢ Abnormalities in the number of sex chromosomes are
relatively common.
➢ The most frequent ones are Klinefelter syndrome and
Turner syndrome.
No disjonction (ND) in the second division
Gametes
Gamete disomic nullosomic normal normal
Zygote trisomy monosomy normal normal
Non-disjunction during the 2nd meiotic division of spermatogenesis
Anomalies de Structure
Factors that can alter the structure of chromosomes: X-rays, atomic radiation, various
chemical agents.
Rearrangements affecting one chromosome Rearrangements affecting more than 2 chromosomes
➢ Inversion (inv): Break and reattachment after ➢ Translocation (tr): Exchange of material
inversion. between two non-homologous
chromosomes.
➢Deletion (del): Loss of chromosome fragments.
➢Reciprocal translocation (t): Reciprocal
➢Duplication (dup): Chromosome region
exchange of material between chromosomes.
duplicated.
➢Robertsonian translocation (rob): Involves
➢Ring (r): Circular chromosome. acrocentric chromosomes (13, 14, 15, 21,
➢Isochromosomes (i): Formation of a and 22).
chromosome with two identical arms. ➢Insertion (ins): Insertion of material into a
➢Small supernumerary chromosomes (mar): chromosome.
Additional small chromosomes.
Rearrangements involving 1 chromosome
Terminal Deletion Interstitiel Deletion
Example: Terminal deletion - A break with the loss of the acentric segment resulting in
partial monosomy.
Partial karyotype in a patient with 46,XY,del(4p)
exhibiting facial dysmorphia, a polymalformative
syndrome, and mental retardation.
Pericentric inversions Paracentrique Inversions
Exemple : Heterozygous pericentric inversion
After two pregnancies, Mrs. Lambert
gives birth to a newborn who dies a few
days later.
➢ These inversions are balanced rearrangements,
but they lead to difficulties in pairing during
meiosis.
➢ Most often, there is the formation of a pairing
loop.
➢ The occurrence of recombination in the inverted
segment leads to the formation of abnormal
gametes through duplication/deficiency.
Schematic representation of chromosome 6.
Example : Heterozygous paracentric inversion
Parents who consulted a doctor after
trying for a long time to have a child but
without success
Crossing-over" can occur during meiosis and can
lead to unbalanced gametes, resulting in nonviable
unbalanced zygotes
Schematic representation of chromosome 12.
Duplication Ring chromosome.
Chromosome normal Isochromosome
Example: Isochromosome - a chromosome composed of two identical arms.
The isochromosome of the long arm of the X chromosome: 46,X,i(Xq) is a common
anomaly in Turner syndrome.
Rearrangements involving multiple chromosomes
Insertion
FISH Insertion
Reciprocal translocation Robertsonian translocation
Reciprocal translocation
Karyotype FISH
46,XY,t(5;20)(q11.2;p13)
Example: Balanced reciprocal translocation between
chromosome 7 and 19t(7;19)(q22;q13.1) Segment (7q22 - 7qter)
exchanged with segment (19q13.1 - 19qter)
Robertsonian translocations
Involves acrocentric chromosomes
(13, 14, 15, 21, and 22)
• Fusion of chromosomes with loss
of short arms: without direct
consequences on carriers
Rob héteroz → phénot
normal
BUT
Offspring → High risk of
Down syndrome
When it comes to a
Robertsonian translocation, the
production of gametes is
disrupted:
• Gametes with trisomy
• Gametes with monosomy Anormal Anormal Anormal Anormal
• Balanced gametes
• Normal gametes
Equilibré Normal
