CYTOGENETICS
CHROMOSOME DISCOVERY AND FLEMMING, BOVERI, AND SUTTON CONNECT
CHROMOSOME STRUCTURE
DISCOVERY OF THE CHROMOSOME
• Mendelian “Factors”
o 1903, rediscovered by Theodore Boveri and
Walter Sutton.
o Eventually called Chromosome Theory of
Inheritance.
• Discovery of sex chromosomes and association
between specific genes and specific chromosomes.
o 1910: Thomas Hunt Morgan
o 1916: Calvin Bridges
DEVELOPING THE CHROMOSOME THEORY
• 1860s: Gregor Mendel and Charles Darwin
o Began to explore possible mechanisms of
heredity.
• Walther Flemming, Theodor Boveri, and Walter
Sutton
o Made a series of significant discoveries
involving chromosomes.
§ What these structures looked like.
§ How they moved during mitosis.
§ What role they likely played in the
transmission of genetic
characteristics.
• Early 20th Century: Thomas Hunt Morgan
o Researchers were finally able to directly link
the inheritance of genetic traits to the
behavior of chromosomes, thereby providing
concrete evidence for what became known
as the Chromosome Theory of Heredity.
EARLY BREAKTHROUGHS: MENDEL AND DARWIN
PROPOSE MECHANISMS OF HEREDITY
• 1865 was marked by two profound biological
breakthroughs:
o Mendel's publication of his experiments in
plant hybridization.
o Darwin's provisional hypothesis
of pangenesis.
• Mendel speculated that cells contained some type of
factor that carried traits from one generation to the
next.
• Darwin proposed that traits could be passed down via
units he termed “gemmules,” which he believed
traveled from every body part to the sexual organs,
where they were stored.
• Because these first attempts to explain the
mechanisms of heredity lacked any scientific support,
their profound importance went unrecognized by the
scientific community for decades.
• Mendel and Darwin's work laid the foundation for
formulating a testable, research-based theory of
heredity.
CANO
CHROMOSOMES TO HEREDITY
• Theodor Boveri
o Began to shed light on the events that take
place in the nucleus during cell division and
their implications.
• Some thirty-five years later:
o Significance of Mendel's work was
emphasized by Walter Sutton.
o Observations of chromosome behavior
during cell division and gamete formation
were consistent with Mendel's findings.
o Basis for chromosome theory and the field
of cytogenetics was created.
WALTHER FLEMMING DESCRIBES CHROMOSOMES
• Middle of 19th Century
o Scientists understood that cells derived from
other cells and that the hereditary
information was located in the nucleus.
o Physical nature of the hereditary material
remained unknown.
o Microscopes of the time provided very poor
resolution of living cellular structures,
making it necessary for investigators to treat
fixed cells with various stains to enhance the
contrast of their contents.
• Walther Flemming
o German anatomist.
o Innovative microscopy techniques and
painstaking precision.
o Chromatin or “Stainable Material”
§ Fibrous network within the nucleus.
§ Coined a few years later by
Heinrich Waldeyer.
o Mitosen
§ Greek word for thread.
§ During cell division, the chromatin
formed threadlike bodies.
o Correctly deduced the sequence of
chromosome movements during mitosis.
§ Based on many observations of
cells in various stages of division.
§ Confirmed decades later by
microscopy of live dividing cells.
o Made the important observation that
chromosomes split along their length during
mitosis.
o Correctly hypothesized that the split
chromosomes were partitioned into different
daughter cells at the end of mitosis.
o Recognized that chromosomal movement
during mitosis offered a mechanism for the
precise distribution of nuclear material
during cell division.
o Provided an invaluable description of the
initial mechanisms underlying the process of
cell division.
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CYTOGENETICS
o Helped paved the way for the discovery of
hereditary mechanisms.
THEORDOR BOVERI LINKS CHROMOSOMES
AND HEREDITY
• End of the 19th Century
o Marked by advancements in cytological
techniques and microscopy.
• Theodor Boveri
o German embryologist.
o Took Flemming's findings to the next level
by providing the first evidence that
chromosomes of germ cell lineage provide
continuity between generations.
o Found evidence for this hypothesis through
his research of early development in the
roundworm species Ascaris
megalocephala (now known as Parascaris
equorum).
o Ascaris embryos provided an excellent
experimental model for Boveri's
observations:
§ Large, clear cells of
the Ascaris embryo have only two
pairs of chromosomes.
§ Embryos develop distinct somatic
cell and germ cell lineages during
the first few cleavage divisions.
o Able to trace the fate of the chromosomes in
individual cell lineages with great precision.
o Made surprising observation that the full
complement of chromosomes was retained
only in the Ascaris germ cell lineage, the
source of future gametes.
o Chromosomes in somatic cells underwent a
curious process of fragmentation and
elimination known as chromosome
diminution.
§ Chromosome diminution does not
occur in mammals.
o Recognized that chromosome number was
reduced in the gametes.
o In Ascaris eggs:
§ Meiosis does not
occur until fertilization is complete.
§ Able to observe the behavior
of egg and sperm chromosomes
following fertilization.
o Noted that Ascaris eggs retained only two
chromosomes after the polar body formed,
and that the normal number of four
chromosomes was restored following fusion
of the sperm and egg pronuclei.
o Provided one of the first descriptions of
meiosis.
WALTER SUTTON FINDS EVIDENCE FOR
MENDEL’S PRINCIPLES
• Walter Sutton
CANO
o Near the dawn of 20th Century.
o Working independently.
o American graduate student.
o Kansas farm boy.
o Confirmed and expanded upon Boveri's
observations using a superior cytological
model.
o Made the serendipitous discovery that it was
possible to distinguish individual
chromosomes in cells undergoing meiosis in
the testes of the lubber
grasshopper, Brachystola magna.
o Classic paper published in 1902:
§ Described the configurations of
individual chromosomes in cells at
various stages of meiosis.
o Able to identify:
§ 11 pairs of chromosomes that could
be distinguished by their sizes.
§ Accessory singleton presumed to
be a sex chromosome.
• Apparent lack of a partner.
• Sorted into only half of the
sperm cells.
• Remaining half never
received a copy.
o Though the accessory chromosome was
unpaired, it still replicated and entered
stages of mitosis in the same manner as all
other chromosomes = “true chromosome”
and not simply an accessory.
o Postulated that all chromosomes have a
stable structure, or “individuality”.
§ Maintained between generations.
§ Used this property to follow the
behavior of individual
chromosomes through the various
stages of meiosis,
including synapsis.
o Recognized that his observations were
consistent with Mendel’s whose findings
had only recently been rediscovered.
§ Closed his 1902 paper with the
statement, “I may finally call
attention to the probability that the
association of paternal and
maternal chromosomes in pairs and
their subsequent separation during
the reducing division as indicated
above may constitute the physical
basis of the Mendelian law of
heredity.”
§ Articulated the chromosomal
theory of inheritance.
o Went on to explain the basis for the ongoing
variation among heritable traits.
o Position of each chromosome at the midline
during metaphase was random.
o There was never a consistent maternal or
paternal side of the cell division.
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CYTOGENETICS
o Each chromosome was, therefore,
independent of the others.
o When they separated into gametes, the set of
chromosomes in each daughter cell could
contain a mixture of the parental traits, but
not necessarily the same mixture as that of
other daughter cells.
o Newly discovered chromosomal
independence during meiosis meant that the
number of possible chromosomal
combinations for each gamete could be
calculated based on the number of
chromosomes in the organism.
o Specifically, there are 2n possible
combinations of chromosomes in gametes.
§ “n” = number of chromosomes in
the gamete.
o Considering all the possible pairings of one
gamete with another, the variation in zygotes
is (2n)2, which results in some fairly large
numbers.
o Provided examples of the potential variation
among hypothetical organisms with gamete
chromosome numbers ranging from 1 to 19.
o Correctly assumed that there was more than
one trait present on each chromosome, so
the actual total variation was even higher
than any of those included in the table.
o This observation of potential variation added
strength to Sutton’s assertion that he had
discovered the physical basis for Mendel's
principle of independent assortment.
o Still lacking was definite proof.
o Scientists needed an experimental system in
which the inheritance of genetic traits could
be linked directly to the behavior of
chromosomes.
§ Distinct mutation in the fruit
fly Drosophila melanogaster.
THOMAS HUNT MORGAN EXPERIMENTALLY
DEMONSTRATES CHROMOSOME THEORY
• Early years of the 20th Century
CANO
o Thomas Hunt Morgan and his colleagues at
Columbia University identified hundreds
of Drosophila genes and made many pivotal
discoveries about genetic transmission.
o Cytological examination showed
that Drosophila possesses four pairs of
chromosomes, including a pair of sex
chromosomes.
§ Female = two identical X
chromosomes.
§ Males = single X chromosome and
smaller gene-poor Y chromosome.
o Sex in fruit flies is determined by the
number of X chromosomes, rather than by
the presence of the Y chromosome.
• One day, Morgan’s associates discovered a male fly
with unusual white eyes in their cultures.
o Breeding experiments quickly established
that the white eye color was caused by
a recessive mutation in the gene responsible
for normal red eye color.
o Gene was probably located on the X
chromosome.
o Chromosome Theory:
§ Male flies would always display the
eye color encoded on their single X
chromosome.
§ Female flies would develop white
eye color only when they inherited
two mutant versions of the eye
color gene.
o Rare occasions:
§ “Exceptional” white-eyed females
among the progeny of a
heterozygous female fly and a
normal male.
• Lilian Vaughn Morgan
o Thomas Morgan’s wife.
o Had the insight to recognize that these
“exceptional” females might have an
unusual chromosome composition.
o Females had two X chromosomes as well as
a Y chromosome.
§ XXY females resulted from defects
in meiosis that caused a high
frequency of nondisjunction.
§ Failure of chromatids to separate
during the second meiotic division.
o When eggs containing two nondisjoined X
chromosomes, each of which carried the
mutant white gene, were fertilized by a Y-
bearing sperm, the product was an XXY
female with white eyes.
o Rather than disproving the chromosome
theory, these “exceptional” females actually
provided strong experimental support that
genes were in fact located on chromosomes.
WHAT IS A CHROMOSOME?
• Rod shaped filamentous structures.
DANICA YAMBAO 2OMT
CYTOGENETICS CANO
• Threadlike structures or “colored bodies” DNA COILING
o Chroma = color; strong affinity to dyes.
o Soma = body
• Made of protein and a single molecule of DNA
• Factors that distinguish one species from another
• Different living organisms have various number of
chromosomes.
• Only visible during cell division using a light
microscope.
• Not visible in active nucleus due to their high water
content.

WHAT DO CHROMOSOMES DO?

• Enable transmission of genetic information from one
generation to the next.
• In Mitosis
o Ensure daughter cell retains its own
complete genetic complement.
• In Meiosis
o Enable each mature ovum and sperm to
contain a unique single set of parental genes.
• Vehicle that facilitates reproduction and maintenance
of species.

HUMAN CHROMOSOMES
• 44 autosomes
• 2 sex chromosomes
o XX (female)
o XY (male)
• 23 pairs • To package all of this DNA into a tiny cell nucleus,
• Extra-chromosomal DNA it is coiled at several levels.
o Other DNA materials found in • First, the DNA is wound around a histone protein
mitochondria. core to form a nucleosome.
• About 140 to 150 DNA bases are wound around
each histone core.
• 20 to 60 bases form a spacer element before the
next nucleosome complex.
• Nucleosomes in turn form a helical solenoid;
each turn of the solenoid includes about six
nucleosomes.
• Solenoids themselves are organized into chromatin
loops, which are attached to a protein scaffold.
• Each of these loops contains approximately 100,000
base pairs (bp), or 100 kilobases (kb), of DNA.
• End result of this coiling and looping is that the
DNA, at its maximum stage of condensation, is
only about 1/10,000 as long as it would be if it were
fully stretched out.

METAPHASE CHROMOSOMES
• Replicated condensed chromosome with sister
chromatids.
• Can be counted easily during the mitotic metaphase
stage.

DANICA YAMBAO 2OMT

CYTOGENETICS
• The mechanism for packaging this DNA in the
nucleus is critical to maintaining its integrity and
organization.
• Packaging also influences gene expression.
• Basic element of chromosome structure is the
nucleosome.
o Repeating unit composed of double-stranded
DNA wrapped almost twice around a
complex of histone proteins.
o Compacts DNA by reducing its length about
7-fold.
o Nucleosome’s protein core
§ Composed of two molecules of
each of four different histone
proteins, H2A, H2B, H3, and H4.
§ Contain a large number of lysine
and arginine amino acids, making
the protein core highly basic.
§ This helps it bind to the negatively-
charged phosphate groups in DNA.
• Short unbound stretch of linker DNA is found
between consecutive nucleosomes.
• A fifth histone, H1, binds to the linker DNA and may
help connect adjacent nucleosomes during early
chromosome compaction, although there are
competing models of the resulting 30-nm fiber.
o This shortens DNA length another 7-fold, to
a total of almost 50-fold over the initial
DNA molecular length.
• In addition to these histone proteins, there is a large
non- histone protein component in a chromosome.
CANO
o Highly diverse.
o Largest class is the transcription factors that
regulate gene expression.
o Other examples are the non-histone proteins
in the centromeric kinetochores that function
in chromosome movement during cell
division and the structural proteins of the
nuclear matrix and supporting scaffold of
condensed chromosomes.
• At a third level of compaction, the 30-nm fibers are
hypothesized to attach as radial loops to filaments of
the dynamic network of proteins that make up the
nuclear matrix.
o These looped domains of between 25,000 bp
and 200,000 bp in size are anchored to the
matrix filaments by other kinds of non-
histone proteins.
• Matrix-attachment regions (MARs) or scaffold-
attachment regions (SARs) are dispersed at intervals
throughout the genome.
o This results in a further 200- to 250-fold
shortening of the chromosomes to a total of
about 10,000-fold above the naked DNA.
CHROMOSOME SIZE
• In contrast to other cell organelles, the size of
chromosomes shows a remarkable variation
depending upon the stages of cell division.
• Interphase:
o Chromosome are largest and thinnest.
• Prophase:
o Progressive decrease in length.
o Increase in thickness.
• Anaphase:
o Chromosomes are smallest.
• Metaphase:
o Chromosomes are the most easily observed
and studied during metaphase when they are
very thick, quite short, and well spread in
the cell.
MORPHOLOGY OF CHROMOSOME
• Chromatids
o Two identical strands which are the result of
DNA replication.
o Sister chromatid = 1 chromosome.
• Centromere
o Central region.
o Region where two sister chromatids of a
chromosome appear to be joined or held
together.
o Also termed as Primary Constriction where
sister chromatids are linked.
o Consists of several hundred kilobases of
repetitive DNA.
o Responsible for chromosome movement at
cell division.
o When chromosomes are stained:
§ dark stained region = centromere.
DANICA YAMBAO 2OMT
CYTOGENETICS CANO
o During mitosis, the centromere that is shared CHROMOSOME TYPES
by the sister chromatids must divide so that • Based on centromere position:
the chromatids can migrate to opposite poles o Metacentric
of the cell. § Middle.
o During the Meiosis I, the centromere of SC § Yielding arms of roughly equal
remain intact. length.
o During the Meiosis II, the centromere act § Centromere is centrally located.
like in the mitosis. § 5 pairs in humans.
o Important structure for chromosome o Submetacentric
segregation. § Off-center centromere.
o Divides the chromosome into: § “q” arm is longer.
§ Short arm: designated as p (petite) § Unequal length of chromosome
§ Long arm: designated as q (queues arms.
or “g” = grande) o Acrocentric
§ Acro = peak.
§ Very close to one end.
§ Yielding a small short arm.
§ Often associated with small pieces
of DNA called satellites, encoding
rRNA.
§ 5 pairs in humans.
o Telocentric
§ V-shaped.
§ Centromeres at the terminal end.
§ Not found in humans.
§ Common in animals (house
mouse).

• Kinetochore
o Organelle located at the centromere region.
o Actual location where the attachment occur.
o Composed of both DNA and protein.
o DNA sequence within this region is called
the CEN DNA.
o Acts as the microtubule organizing center.
o Facilitates spindle formation by
polymerization of tubulin dimers to form
microtubules in early mitosis.

• Based on arms ratio:

o 1964, Levan et al.
o Determine the ratio of p and q arms for each
chromosome type.
o Important in chromosomal aberrations.
DANICA YAMBAO 2OMT
CYTOGENETICS CANO
• Comes from the Greek words for color (chroma) and
body (soma) = strongly stained by some colorful dyes
used in research.

WHAT DO CHROMOSOMES DO?

• Keeps DNA tightly wrapped around spool-like
proteins, called histones.
o Without such packaging, DNA molecules
o Subtelocentric would be too long to fit inside cells.
§ Centromere closer to the end than • Key part of the process that ensures DNA is
to the center. accurately copied and distributed in the vast majority
§ Mas malapit sa center yung of cell divisions.
submetacentric. • Changes in the number or structure of chromosomes
in new cells may lead to serious problems
• Reproductive cells containing the right number of
chromosomes and have the correct structure is
crucial.
o If not, the resulting offspring may fail to
develop properly.

DO ALL LIVING THINGS HAVE THE SAME TYPES OF

CHROMOSOMES?
• Chromosomes vary in number and shape among
living things.
o Most bacteria: one or two circular
chromosomes.
o Humans, animals and plants: linear
chromosomes that are arranged in pairs
within the nucleus of the cell.
• Telomere • Reproductive Cells or Gametes
o Tip/end of each chromosome. o Only human cells that do not contain pairs
o Forms a T-loop. of chromosomes.
o Tandem repeats of the hexameric sequence o Carry just one copy of each chromosome.
‘TTAGGG’. o When two reproductive cells unite, they
o Required for the replication and stability of become a single cell that contains two copies
the chromosome. of each chromosome.
o Functions in preserving chromosome o Cell divides and its successors divide
stability: numerous times = mature individual with a
§ Preventing abnormal end-to-end full set of paired chromosomes in virtually
fusion of chromosomes. all of its cells.
§ Protecting the ends of • Circular chromosome
chromosomes from degradation. o Found in mitochondria.
§ Ensuring complete DNA o Structures located outside the nucleus that
replication. serve as the cell’s powerhouses.
§ Having a role in chromosome o In the past:
pairing during meiosis. § Mitochondria were free-living
bacteria with the ability to convert
CHROMOSOMES FACT SHEET oxygen into energy.
§ When these bacteria invaded cells
WHAT IS A CHROMOSOME? lacking the power to tap into
• Thread-like structures located inside the nucleus of oxygen’s power, the cells retained
animal and plant cells. them, and, over time, the bacteria
• Made of protein and a single molecule of evolved into modern-day
deoxyribonucleic acid (DNA). mitochondria.
• Passed from parents to offspring, DNA contains the
specific instructions that make each type of living WHAT ARE CENTROMERES?
creature unique. • Constricted region of linear chromosomes.
• Help to keep chromosomes properly aligned during
the complex process of cell division.
DANICA YAMBAO 2OMT
CYTOGENETICS
• As chromosomes are copied in preparation for
production of a new cell, the centromere serves as an
attachment site for the two halves of each replicated
chromosome, known as sister chromatids.
WHAT ARE TELOMERES?
• Repetitive stretches of DNA located at the ends of
linear chromosomes.
• Protect the ends of chromosomes in a manner similar
to the way the tips of shoelaces keep them from
unraveling.
• Lose a bit of their DNA every time a cell divides.
o When all of the telomere DNA is gone, the
cell cannot replicate and dies.
• White blood cells and other cell types with the
capacity to divide very frequently have a special
enzyme that prevents their chromosomes from losing
their telomeres = generally live longer than other
cells.
• Play a role in cancer.
o Chromosomes of malignant cells usually do
not lose their telomeres.
o Fuel the uncontrolled growth that makes
cancer so devastating.
HOW MANY CHROMOSOMES DO HUMANS HAVE?
• Humans = 23 pairs of chromosomes = 46.
• Fruit fly = 4 pairs.
• Rice plant =12 pairs.
• Dog = 39 pairs.
HOW ARE CHROMOSOMES INHERITED?
• One copy of each chromosome is inherited from the
female parent and the other from the male parent.
• Pattern of inheritance is different for the small
circular chromosome found in mitochondria.
o Only egg cells keep their mitochondria
during fertilization.
o Mitochondrial DNA is always inherited
from the female parent.
o Humans: some forms of hearing impairment
and diabetes have been associated with
DNA found in the mitochondria.
DO MALES HAVE DIFFERENT CHROMOSOMES
THAN FEMALES?
• Differ in sex chromosomes:
o Females = two X chromosomes.
o Males = one X and one Y chromosome.
• Inheriting too many or not enough copies of sex
chromosomes can lead to serious problems.
o Females who have extra copies of the X
chromosome:
§ Usually taller than average.
§ Some have mental retardation.
o Males with more than one X chromosome:
§ Klinefelter Syndrome
§ Tall stature.
§ Impaired fertility.
CANO
o Women with only one X chromosome.
§ Turner Syndrome
§ Very short.
§ Usually do not undergo puberty.
§ some may have kidney or heart
problems.
HOW WERE CHROMOSOMES DISCOVERED?
• Late 1800s
o First observed chromosomes.
o Nature and function of these cell structures
were unclear.
• Early 1900s
o Researchers gained a much better
understanding of chromosomes through
Thomas Hunt Morgan’s pioneering studies.
o Made the link between chromosomes and
inherited traits by demonstrating that the X
chromosome is related to gender and eye
color in fruit flies.
CHROMOSOMAL ABERRATIONS
• Aberrations = abnormality.
• Abnormal structure or number of chromosomes.
• Chromosomal abnormalities represent changes in
chromosomes (46 in human somatic cells) or their
structural modifications.
• Certain genomic mutations explain chromosomal
number abnormalities, while there are chromosomal
aberrations that explain chromosomal structure
abnormalities.
• Chromosomal disorders form a category of human
genetic disease that are manifested by developmental
and reproductive abnormalities, as well as playing an
important role in the pathogenesis of malignancy.
• Depending on the nature of aberration, it can lead to
severe birth defect or defects incompatible with life.
• Diagnosis: by doing karyotyping.
• Chromosomal abnormalities may be produced by:
o Mitosis deregulating factors that produce
DNA tears or affects replication
o Chemical factors
§ Teratogen: agent or factor which
causes malformation of an embryo.
o Physical factors (e.g. ionizing radiation)
o Biological factors (e.g. viruses)
o Mother advanced age
§ Increased aneuploidy risk.
o One of the parents is carrier (e.g.
translocation)
o Chromosomal rearrangements (e.g. unequal
crossing-over – nondisjunction of
chromosomes)
• What clinical presentations would lead one to suspect
a chromosomal abnormality?
o Infertility
§ Not being able to get pregnant
despite having frequent unprotected
sex for at least 1 year.
DANICA YAMBAO 2OMT
CYTOGENETICS CANO
§ Not always a direct result of a o Part of a chromosome breaks off and
medical procedure. attaches to another nonhomologous
o Sterility chromosome.
§ Inability to create offspring because
of a certain procedure.
§ Vasectomy, tubal ligation,
hysterectomy.
o Intersexes
§ Hermaphrodites
• Both ovaries and testes are
present.
§ Pseudohermaphrodites
• Born with one
chromosomal or gonadal
sex but he or she has
developed some secondary
characteristics of the other.
o Multiple Congenital Malformations STRUCTURAL ABERRATIONS
• Ways by which a chromosome can be structurally
atypical:
o Too much or too little genetic material
§ Balanced
• If the amount of genetic
material is normal.
• All the genetic
information is present in
the right amount but it is
placed in a wrong location
or different order.
• Usually does not lead to
phenotypic abnormality
(hindi masyado nakikita).
§ Unbalanced
• If there is an excess or a
deficient amount of
genetic material or DNA
content.
• Lost or gain of a
o Mental Retardation chromosome content.
• Variations in the chromosome number • Phenotype of carrier is
o Aneuploidy abnormal.
§ Addition or loss of one or more o Stretch of DNA is inverted or moved and
chromosomes. stuck onto a different type of chromosomes.
§ Trisomy (2n + 1) § Chromosome structure
§ Monosomy (2n – 1) chromosomes variations may result
o Polyploidy from chromosome breakage.
§ Addition of chromosome sets. § Broken piece of chromosomes tend
§ Triploidy (3n) to re-join; if there is more than one
§ Tetraploidy (4n) break, rejoining usually occurs at
• Alterations in the chromosome structure random and not necessarily with
o Deletion the correct ends.
§ Lost a part of a chromosome. • Four common types:
o Duplication o Deletion or Deficiency
§ Segment of a chromosome is o Duplication or Repeat
repeated. o Inversion
o Inversion o Translocation
§ Part of a chromosome is oriented in • Example:
the reverse of its usual direction. o Consider a normal chromosome with genes
o Reciprocal Translocation in alphabetical order: a b c d e f g h i
DANICA YAMBAO 2OMT
CYTOGENETICS CANO
o Deletion: part of the chromosome has be o Majority of deficiencies detected are
removed: a b c g h i intercalary type within the chromosomes.
o Duplication: part of the chromosome is • Generally produce striking genetic and physiological
duplicated: a b c d e f d e f g h i effects.
o Inversion: part of the chromosome has been • Homozygous
reinserted in reverse order: a b c f e d g h i o Lethal because most genes are necessary for
o Translocation: parts of two non-homologous life.
chromosomes are joined. o Zero copies.
§ If one normal chromosome is a b c o Resulting in zygotic loss, still births or
d e f g h i and the other infant deaths.
chromosome is u v w x y z, then a • Heterozygous
translocation between them would o Genes on the normal homologue are
be a b c d e f x y z and u v w g h i. hemizygous.
o Only one copy.
• Crossing-over is absent in deleted region of a
chromosome since this region is present in only one
copy in deletion heterozygotes.

DELETION OR DEFICIENCY
• Characterized by chromosomal fragment lost.
• Loss of a chromosome segment.
• First structural aberration detected by Bridges in
1917 from his genetic studies on X chromosome of CRI-DU-CHAT (CAT CRY SYNDROME)
Drosophila. • Other names:
• Terminal Deletion o Lejeune’s syndrome
o Produced by one point chromosomal tearing o Chromosome 5p deletion syndrome
followed by terminal fragment lost. o 5p monosomy
• Interstitial/Intercalary Deletion o 5p minus syndrome
o Produced by chromosomal tearing in two o 46,XX5p- or 46,XY5p-
points followed by intercalary fragment lost. • Identified by Jérôme Lejeune in 1963.
• Rare genetic syndrome due to chromosome deletion
on chromosome 5.
• Chromosome deficiency is in the short arm of
chromosome 5.
• French term (“cat cry” or “call of the cat”) referring
to the characteristic cat-like cry of affected children.
• Name came from a catlike mewing cry from small
weak infants with the disorder.
• Patients die in infancy or early childhood.
• Clinical Presentation
o Characteristic cat-like cry due to
underdeveloped larynx.
• Can result to terminal deficiency or intercalary o Microcephaly (small head due to
deficiency. underdeveloped brain)
o A single break near the end of the o Round “moon-like” face
chromosome would be expected to result in o Micrognathia
terminal deficiency. § Condition wherein the lower jaw is
o If two breaks occur a section may be deleted undersized.
and an intercalary deficiency created. § Mandibular hypoplasia.
o Terminal deficiencies might seem less § Interferes with breathing and
complicated. eating.

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CYTOGENETICS CANO
o Hypertelorism • Intellectual disability and seizures.
§ Increased distance between organs
or body parts.
§ Orbital Hypertelorism
• Most common.
• Condition wherein the
distance between the inner
eye corners and distance
between the pupil is
greater than normal.
• Other characteristics:
o Broad face and saddle nose
o Physical and mental retardation

• Features:
o Cat-cry
o Feeding problem
o Low BW and poor growth
o Severe cognitive, speech, motor delays
o Behavioral problems (hyperactive, tantrums,
etc.)
o Unusual facial features
§ Hypotonia (low muscle tone)
§ Microcephaly
§ Strabismus (eyes do not align)
§ Low set ears
o Single palmar crease
o Cardiac defects
o Significant intellectual disability
WOLF-HIRSCHHORN SYNDROME
• 4p16.1
• Caused by partial deletion of the short arm of
chromosome 4.
• Distinctive facial features:
o Broad flat nasal bridge and high forehead =
“Greek warrior helmet” appearance.
o Short philtrum (distance between nose and
upper lips) and downturned mouth.
o Micrognathia and poorly formed ears (small
holes/pits or flaps of skin).
o Widely spaced and protruding eyes.
• Delayed growth and development.
JACOBSEN SYNDROME
• Condition caused by a loss of genetic material that
occurs at the end (terminus) of the long (q) arm of
chromosome 11.
• Also known as 11q Terminal Deletion Disorder.
• Delayed development (e.g. speech, motor skills)
• Cognitive impairment and learning difficulties
• Behavioral problems
o Compulsive behavior.
• Attention-deficit/Hyperactivity Disorder (ADHD)
o Short attention span.
• Impaired communication and socialization skills
o Autism Spectrum Disorder
• Hypertelorism
• Ptosis (droopy eyelids)
• Macrocephaly (large head)
• Epicanthal Folds
o Mongoloid folds/Epicanthic folds.
o Skin folds covering the inner corners of the
eyes.
• Trigonocephaly
o Skull abnormality which gives the forehead
pointed appearance.
OTHER SYNDROMES DUE TO DELETION
• Prader-Willi Syndrome
o 15q11−13
o Discovered by Andrea Prader and Heinrich
Willi.
o Neonatal hypotonia
o Initial feeding difficulties
§ As they age = hyperphagia
(overeating, constantly hungry) =
leads to obesity and type II diabetes
mellitus.
o Obesity of face, trunk, and limbs (after first
year of life)
o Prominent forehead
o Almond-shaped eyes
o Triangular upper lips (thin upper lips)
o IQ 20-80
o Short stature
o Small hands and feet
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o Hypoplasia of external genitalia
o Tendency of diabetes mellitus

• Angelman Syndrome *Dolichocephalic: head is longer than what is expected

o 15q11−13 relative to its width.
o “Happy Puppet Syndrome” = because of *Remember: mahilig magsuck si Angel!
happy appearance and hypertonia.
o Hypertonia DUPLICATION
§ Increased muscle tone. • Presence of an additional chromosome segment, as
§ Stiff = difficulty to walk. compared to that normally present in a nucleus.
o Ataxic gait • In a diploid organism, presence of a chromosome
§ Unsteady walk. segment in more than two copies per nucleus is called
§ Nauuna yung heels before yung duplication.
toes.
o Characteristic arm posture
o Prominent jaw
o Deep set eyes
o Happy appearance
o Laughter
o Absent speech
o Learning disability
• Four types:
o Tandem Duplication
§ Extra chromosome segment maybe
located immediately after the
normal segment in a precisely same
orientation.
o Reverse Tandem Duplication
§ Gene sequence in the extra segment
of a tandem is in the reverse order.
o Displaced Duplication
§ Extra segment is located in the
same chromosome but away from
the normal segment.
o Translocation Duplication
§ Addition chromosome segment that
is located in a nonhomologous
*UPD = Uniparental Disomy: two copies of chromosome chromosome.
come from same parents.
*UBE3A = Ubiquitin Protein Ligase E3A

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CHARCOT-MARIE-TOOTH DISEASE TYPE 1A
• Extra copy of the PMP22 gene on chromosome 17.
o PMP22: Peripheral Myelin Protein 22
• Autosomal dominant.
• Affects the peripheral nerves.
• Experience weakness and atrophy of the muscles in
the lower leg during adolescence.
• Sensory loss.
UNEQUAL CROSSING-OVER
• Duplication of the band 16A of X chromosome of
Drosophila produces Bar eye.
• Believed to originate due to unequal crossing over
between the two normal x chromosome.
CANO
ISOCHROMOSOME
• Duplication of one arm but lacks the other.
• Deletion of one and duplication of the other.
• Abnormal chromosome formed by two short arm or
two long arms.
• Breakage of a chromatid with fusion above the
centromere or transverse division.
• Transverse division or transversal cleavage of the
centromere.
• Isochromosomes of most autosomes are lethal, but
those of the X chromosome [46,X,I(Xq)] can yield a
phenotype similar to that seen in Turner syndrome.
RING CHROMOSOME
• Result from breaks near both telomeres of a
chromosome, which aberrantly repair to form a ring,
with the regions distal to the breaks being lost.
• If a ring has a centromere, it will be passed down to
generations in mitosis.
• Appear after chromosomal tearing in two points of
different shoulders followed by terminal segment loss
and the joining of the ends to form a ring structure.
INVERSION
• When a segment of chromosome is oriented in the
reverse direction.
• First detected by Strutevant and Plunkett in 1926.
• Occur when parts of chromosomes become detached,
turn through 180º, and are reinserted in such a way
that the genes are in reversed order.
• Classified into two types:
o Paracentric Inversion
§ Does not include centromere.
o Pericentric Inversion
§ Includes centromere.
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exchange of chromosome section between
them.
o Segments reciprocally transferred.
o Most frequent.
o Best studied.

TRANSLOCATION
• When a portion of one chromosome is transferred to
another chromosome.
• Integration of a chromosome segment into a
nonhomologous chromosome.

THE PHILADELPHIA CHROMOSOME

• Also called Ph1.
• Results in one chromosome 9 longer than the normal
and one chromosome 22 shorter than the normal.
• This translocation is designated t(9;22).
• Causes chronic myelogenous leukemia.
THREE TYPES OF TRANSLOCATION • BCR ABL1: fusion gene.
o BCR
• Simple Translocation
§ Breakpoint Cluster Region Protein
o Involve a single break in the chromosomes
§ Chromosome 22
and the transfer of broken piece of this
o ABL1
chromosome to the end of another.
§ Abelson Murine Leukemia Viral
o Rare.
Oncogene Homolog 1
o A terminal segment of chromosome is
§ Chromosome 9
integrated at one end of a nonhomologous
o Highly sensitive test for chronic
region.
myelogenous leukemia.
• Shift
o 95% of the cases are positive for BCR-
o Involve three breaks and the transfer of a
ABL1 gene.
two break section of one chromosome
• Chromosome abnormality not found in any
within the break produced in a
nonleukemic white blood cells, nor in any other cells
nonhomologous chromosome.
of the patient’s body.
o An intercalary segment is integrated with a
nonhomologous chromosome.
• Reciprocal Translocation or Interchanges
o Produced by a single breaks in two
homologous chromosomes, followed by an
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ROBERTSONIAN TRANSLOCATION
• Down Syndrome Carrier: t(14;21)
• Produced by two acrocentric chromosomes pairing at
the centromere followed by a long arm fusion and a
short arm lost.
• A portion of, or an entire, chromosome 21 fuses with
one chromosome 14.
• Individual is phenotypically normal, but could have
children with Down Syndrome.
o Gametes may be produced that contain one
21 and the abnormal 14 (fused with 21).
o If fertilized, these would lead to Down
Syndrome.
• Only occur in chromosome 13, 14, 15, 21, and 22.

• Reciprocal translocation = t(9;22)

• The fusion of the two genes (red and green dots) in
the Philadelphia chromosome is what eventually
causes the leukemia.
• This is because it forms a new protein that causes
uncontrollable cell division of cells in the bone PLOIDY
marrow that give rise to WBC. • Basic chromosome set or the exact multiples of that
set represented by n.
• Euploid
o A somatic cell with the normal number of
chromosomes for that species.
o Organisms with one complete set of
chromosomes.
o Applies to haploid and diploid organisms.
• Polyploid
o A cell with one or more extra sets of
chromosomes.
o Group.
• Aneuploidy
o Presence of additional chromosome or
missing individual chromosomes.

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o Variation in the number of individual § Trisomy
chromosomes but not the total number of • One additional.
sets of chromosomes. • (23x2) +1 = 47
o The discovery of aneuploidy dates back to § Monosomy
1916 when Bridges discovered XO male and • One less.
XXY female Drosophila which had 7 and 9 • (23x2) – 1 = 45
chromosomes respectively instead of normal
8.

• Syndromes resulting from Aneuploidy:

o Trisomy 13
o Trisomy 18
o Down Syndrome
o Turner Syndrome
o Klinefelter Syndrome
o XYY Syndrome

MECHANISMS LEADING TO NUMERICAL

CHROMOSOMAL DISORDERS
• Trisomies may result from the failure of separation
(“non-disjunction”) of homologous chromosomes at
meiosis I or from failure of separation of chromatids
in meiosis II.
o Non-disjunction: chromosomal or chromatid
• Nullisomic segregation error during cell division.
o Loss of one chromosome homologous pair. • Monosomies may result from non-disjunction or from
• Monosomic anaphase lag (delay in the movement of one
o Loss of single chromosome. chromosome from the metaphase plate during
• Double Monosomic anaphase, resulting in the loss of the chromosome).
• Trisomic • Polyploidy arises as a result of fertilization by two
o Extra chromosome. sperm or ovum due to failure in meiosis.
• Tetrasomic
o Extra chromosome pair.
• Double Tetrasomic
• Chromosomal Variations
o Polyploidy
§ Triploidy
• Three sets.
• 23x3 = 69.
§ Tetraploidy
• Four sets.
• 23x4 = 92.
o Aneuploidy

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DOWN SYNDROME (TRISOMY 21/TRISOMY G) o Single Palmar Fold
• Best known and most common chromosomes related o Protruding Tongue
syndrome. o Macroglossia
• Formerly known as “Mongolism:. § Large tongue.
• In 1866, physician John Langdon Down published an o Short neck
essay in England in which he described a set of o Brushfield spots
children with common features who were distinct § Small white or grayish-white spots
from other children with mental retardation he on the periphery of the iris.
referred to as “Mongoloids”. o Congenital heart defects
• One child in every 800-1000 births has Down o Excessive space between large and 2nd toes
Syndrome. o Broad head
o 40 years old: 1 in 100 births. o Very round face
o 45 years old: 1 in 40 births. o Mental retardation
• Chromosome 21: smallest human chromosome.
• Trisomy G
o G group refers to small acrocentric
chromosomes which includes 21, 22, and Y.
• Patients with Down Syndrome:
o Short in stature (four feet tall)
o Epicanthal fold
o Broad short skulls
o Wild nostrils
o Large tongue
o Stubby hands
• Some babies may have:
o Short necks
o Small hands
o Short fingers
• Characterized as low in mentality.
• Results if the extra chromosome is number 21.

PATAU SYNDROME (TRISOMY 13)

• Three copies of chromosome 13.
• Trisomy D: group D
• Chromosome nomenclature: 47, +13
• Chromosome formula: 2n+1
• Clinical syndrome: Trisomy-13
• Estimated frequency birth: 1/20,000
• First reported by Klaus Patau.
• Main phenotypic characteristics:
o Mental deficiency
o Deafness
o Minor muscle seizures
o Cleft lip
o Cardiac anomalies
• Features:
• Features of Down Syndrome o Polydactyly or extra fingers
o Microgenia § Most commonly seen.
§ Small or deformed chin. o Microcephaly
o Mongoloid Fold § Undeveloped brain.
o Hypotonia o Incomplete cleavage of the embryonic
§ “Floppy baby syndrome” forebrain/holoprosencephaly
o Flat Nasal Bridge o Scalp skin defects/aplasia cutis

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o Hypertelorism o Ullrich-Turner Syndrome
o Gonadal Dysgenesis
o Monosomy X
o 45 X0 or 45 XO
• Chromosome nomenclature: 45, X
• Chromosome formula: 2n-1
• Clinical syndrome: Turner
• Estimated frequency birth: 1/25,000 Female
• Main phenotypic characteristics:
o Female with retarded sexual development
o Usually sterile
o Short stature
o Webbing of skin in neck region
EDWARDS SYNDROME (TRISOMY 18) o Cardiovascular abnormalities
• Second most common chromosome aberration. o Hearing impairment
• Trisomy E: group E • Features:
• Chromosome nomenclature: 47, +18 o Short stature
• Chromosome formula: 2n+1 o Swelling, broad chest
• Clinical syndrome: Trisomy-18 o Low hair line
• Estimated frequency birth: 1/80,000 o Low set ears
• First described by John Hilton Edwards o Webbed neck
• Main phenotypic characteristics: § Congenital skin fold that runs along
o Multiple congenital malformation of many the sides of the neck down to the
organs shoulders
o Malformed ears § Pterygium colli
o Small mouth and nose with general elf in o Gonadal dysfunction and Amenorrhea
appearance § Incompletely developed ovaries.
o Brown spots (Nevi)
• 90% die in the first 6 months.
o Congenital Heart Disease, Hypothyroidism,
• Features:
Diabetes
o Rocker bottom feet
o Vision and hearing problems
§ Very rigid foot deformity.
o Cognitive deficits
§ Resembles a rocking chair.
o Hypertelorism
o Microcephaly
o Prominent back portion of the head
o Low set malformed ears
o Narrow eyelids
o Widely spaced eyes
o Ptosis
o Underdeveloped nails
o Absence of radius
o Undescended testicles

TURNER SYNDROME
• Monosomy of sex chromosome.
• Only one X present.
• First described by Henry Turner.
• Also called:
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CYTOGENETICS CANO
• Features:
o Infertile male
o Microorchidism
§ Testicular atrophy.
§ Azoospermia: produce semen but
don’t produce functioning sperm.
§ Male reproductive organ is very
small in size.
o Lanky; weak muscles
§ Tall with weak muscles.
o Gynecomastia
§ Breast development.
o Hypogonadism
§ Decrease testicular hormone.
§ Low testosterone level but high
FSH and luteinizing hormone.
o Body similar to female
o More severe:
§ High risk of tumor
§ Breast cancer
§ Osteoporosis
o Trouble using language

• Lymphedema: swelling caused by compromised

lymphatic system.

KLINEFELTER SYNDROME
• Extra X chromosome in males.
• Discovered by Harry Klinefelter.
• Chromosome nomenclature:
o 47, XXY
o 48, XXXY
o 48, XXYY
o 49, XXXXY
o 50, XXXXXY
• Chromosome formula:
o 2n+1
o 2n+2
o 2n+3
o 2n+4
• Clinical syndrome: Klinefelter
• Estimated frequency birth: 1/500 Male
• Main phenotypic characteristics:
o Pitched voice
o Male
o Subfertile with small testes
o Developed breasts
o Feminine
o Long limbs
• Male with female characteristics.
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OTHERS DE LA CHAPELLE SYNDROME
• 47, XXX – Trisomy X Syndrome (Triple X) • XX Male
• 47, XYY – Jacob’s Syndrome • Similar appearance to males with Klinefelter
• XX Male (De la Chapelle Syndrome) Syndrome but:
o Sterile
TRIPLE-X SYNDROME o Usually shorter than average
• 47, XXX – Trisomy X Syndrome o Normal IQ
• Extra X chromosome at pair 23 in females. • Translocation of the portion of the Y chromosome
• Two barr bodies in each cell. containing the (Sex Determining Region Y) SRY
• Taller than average. gene into a X chromosome.
• No unusual physical features. • Features:
• Increased risk of learning disabilities delayed o Abnormal external male genitalia
development of speech and language skills. o Normal male adrenarchal hair pattern
• Weak muscle tone. o Normal intelligence
o Short stature
• Behavioral and emotional difficulties.
o Gynecomastia
o Infertile
o Soft and atrophic testes

TRIPLOIDY
• An individual that has three of every chromosomes,
that is, three haploid set of chromosomes.

JACOB SYNDROME
• 47, XYY (Super Male)
• Extra Y chromosome.
• Not inherited by a parent.
• Occurs at a random event during sperm cell
development.
• Most have normal sexual development and fertility.
• Features:
o Acne
o Aggression
o Tall stature
o Superior muscle strength
• Two Types:
o Reduced muscle coordination
o Molar Triploidy
§ Extra haploid set of chromosomes
of paternal origin.
o Non-molar Triploidy
§ Extra haploid set of chromosomes
of maternal origin.
• Symptoms of Molar Pregnancy
o Uterus expands faster and reaches landmarks
earlier
o More morning sickness
o Earlier signs of Pregnancy Induced
Hypertension (PIH)
o Vaginal bleeding in the 4th month
§ Most common sign.
o Discharge with grape-like vesicles.

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• Process where all chromosomes of an organism are
paired and put in order, to provide a genome wide
idea (“snapshot”) of any individual’s chromosomes.
• Dividing cells are harvested during the metaphase
stage (greatest condensation).
• Laboratory technique that produces an image of an
individual's chromosomes.
• Used to study the changes in chromosome
numbers associated with various aneuploidy
conditions.
PAPP-A: Pregnancy Associated Plasma Protein A • Detect major structural abnormalities of the
B-Hcg: Beta Human Chorionic Gonadotrophin chromosomes.
• Source of diagnostic information for specific birth
NOMENCLATURES USED FOR defects, genetic disorders, and even cancers.
CHROMOSOMAL DISORDERS • Karyotypic analysis is usually considered in the event
• Numerical disorders are described by: of recurrent spontaneous abortion.
o Number of chromosomes o Incidents of major chromosomal
o Sex chromosomes abnormalities are much higher in the first
o + or – chromosome number trimester spontaneously aborted fetuses.
• Structural disorders are described by:
o Number of chromosomes KARYOTYPE
o Sex chromosomes • An individual's collection of chromosomes.
o Mutation (chromosomes involved)
• An organized visual profile of the all chromosomes in
o Break points
the nucleus of a somatic cell of an organism.
o Margins or regions
• Standard chart of chromosomes isolated from a cell at
o P – short arm
metaphase arranged in order by size and structure or
o Q – long arm
physical landmark.
o Examples:
o Group based on sizes and position of
§ Translocation (t)
centromeres.
• [46,XY,t(14,21)(q11;p10)]
§ Inversion (inv)
KARYOTYPING PROCESS
• [46,XY,inv(9)(p12,q14)]
• Preferred sample source
pericentric inversion
o Cells undergoing rapid cell division or
§ Isochromosome (I)
mitosis
• [46,X,I(Xq)] long
§ Bone marrow samples
chromosome arm of X
• Spinal tap.
duplicated
§ Duplication (dup) • First few milliliters are the
best sample for cytogenic
• [46,XY,dup(5)(q20-q30)]
laboratory because they
§ Deletion (del)
contain the highest
• [46,XY,del(15)(q11-q13)]
proportion of cells.
in Prader-Willi Syndrome
• After a the first few
§ Ring Chromosome (r)
milliliters = may blood na
• [46,XX,r(X)(p12,q14)]
which can dilute the
• Based on International Standard Chromosome sample and reduces
Nomenclature (ISCN) number of actively
dividing cells present.
KARYOTYPING • For cancer diagnosis.
• Levitsky § Product of conception i.e. chorionic
o Coined the term “karyotyping”. villus
o Referred to it as the ordered arrangement of • For prenatal diagnosis.
chromosomes. • Occurrence of
o Phenotypical arrangement of chromosomes spontaneous abortion.
only. o Cells that can be stimulated to divide in
• Determines the number and structure of culture
chromosomes in the cell’s nucleus. § Peripheral blood lymphocytes, skin
• Used to detect chromosome aberrations. fibroblasts, and amniocytes
• Involves analysis of the entire chromosome
complement through the use of a microscope.
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CYTOGENETICS CANO
BONE MARROW BIOPSY CHORIONIC VILLUS BIOPSY
• Puncture site: over iliac crest. • Sample of a tissue from the placenta.
• Collected using sterile syringed or vacuum tubes
containing preservatives.
• Transported at room temperature.

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AMNIOCENTESIS
• Procedure in which amniotic fluid is removed from
the uterus.
• Can be performed as early as 10 weeks of gestation.
• 15-30 ml.
• Obtained under sterile conditions.
• Collected in a sterile container.
• Performed earlier than 15 weeks: 1 ml of fluid is
drawn for every week of gestation (3 weeks = 3 ml).
• Use an ultrasound to determine the baby’s exact
location.
•
PREPARATION OF AMNIOCYTES
• Amniotic fluid
o Clear yellow in appearance.
o Brown = prior bleeding in amniotic cavity
which suggests fetal death or miscarriage
o Red/bloody tap = large number of RBCs =
can prevent amniocytes from settling on and
attaching.
CANO
BLOOD LYMPHOCYTES AND SKIN FIBROBLAST
• Samples that can be stimulated to divide in culture.
SPECIMEN COLLECTION
• Green tube for peripheral blood.
KARYOTYPING PROTOCOL
• Theoretically almost all specimens can be used for
chromosomal analysis depending upon the purpose.
o For cancer diagnoses: typical specimens
include tumor biopsies or bone marrow
samples.
o Amniotic fluid or chorionic villus specimens
are used as the source of cells prenatal
diagnosis and for other diagnosis.
o Karyotypes are often performed on a skin
biopsy (i.e. skin fibroblast) and from
peripheral blood specimens, namely
lymphocytes.
• In performing karyotyping:
o Observe proper collection of specimen.
§ Sodium heparin can be used as an
anticoagulant.
o Specimen preparation varies with the type of
tissue undergoing chromosome analysis.
§ In general, the tissues are cultured
in a synthetic media supplemented
with fetal calf serum, antibiotics,
and glutamine to increase the
number of cells in mitosis available
for cytogenetic analysis.
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CYTOGENETICS
§ Culture medium provides nutrients
and amino acids needed for the
growth of the cells.
§ Fetal calf serum/fetal bovine serum
• Contains proteins that
enhances the growth of
cells.
§ Antibiotics
• Suppresses the growth of
contaminants.
§ Glutamine
• Increase the number of
cells in mitosis.
§ For mononuclear cells:
phytohemagglutinin is also added,
which is a known mitogen for
lymphocytes = induces the cells to
undergo mitosis.
o Observe correct time or duration of culture
period before harvesting for chromosome
analysis.
§ Example: blood lymphocytes are
cultured for 48-72 hours while
amniotic fluid cells require 6-14
days.
o Mitotic cells arrested at metaphase.
§ Colcemid/colchicine
• Used to arrest the cells at
metaphase stage of
mitosis.
• Prevents spindle fiber
formation.
o At the end of the culture period, the large
population of dividing cells is treated with a
drug e.g. Colcemid.
o The cells are harvested and treated briefly
with a hypotonic solution (water or 0.7 M
potassium chloride) causing swelling of their
nuclei and separation of chromosomes from
one another.
o The swollen cells are fixed usually using a
combination of methanol and acetic acid,
which preserves the chromosomes
permanently.
o Cells cultured in suspension are dropped
onto a microscope slides, dried and stained
with solutions that would produce various
banding patterns along the chromosome
arms thus each chromosome can to be
unequivocally identified.
o For tissue culture specimen, cells can be
grown attached to the surface of coverslips
or directly on the microscope slide and
processed completely in situ.
§ The in situ method provides a
relatively accurate assessment of
how many independent cells from
the original source have been
analyzed, assuming that each
CANO
colony was derived from one cell
or at most, a few cells.
• Staining of Chromosomes
o The most common staining treatments is G
banding where the cells are first exposed to
an enzyme (trypsin or pancreatin), followed
by treatment with Giemsa stain to produce G
bands.
o The stained metaphase chromosomes
preparations are examined microscopically
with 15-20 cells scanned to identify good
chromosome spread. At least five cells are
fully analyzed and counted.
o Stained slides with good chromosome
spread are then photographed.
KARYOTYPING ANALYSIS
• Like a puzzle, the pictures of the chromosomes are
rearranged to match up pairs and arrange them by
size and structure, from numbers 1 to 22, followed by
the sex chromosomes as the 23rd pair.
• The microphotograph also allow the chromosomes to
be vertically oriented.
o Each chromosome looks like a striped straw.
o It has two arms that differ in length (a short
arm (p) and a long arm (q)), a pinched-in
area between the arms called a centromere,
and a series of light and dark horizontal
bands.
o The length of the arms and the location of
the bands help determine top from bottom.
o In doing a full analysis, each chromosome is
critically compared band-for-band with its
homolog.
• Once the chromosome photo arrangement is
completed, a laboratory specialist evaluates the
chromosome pairs and identifies any abnormalities
that may be present.
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CYTOGENETICS CANO
NORMAL HUMAN KARTOTYPE IDEOGRAM OR KARYOGRAM
• A simple cartoon of a chromosomes, often used in
genomics to show the over-all physical structure of a
chromosomes.
• Provide a pictorial reference point used in or for
localizing the positions of individual genes on
chromosomes, as well as for identifying various
abnormalities associated with a range of
chromosomal disorders.

BANDING TECHNIQUES
• Stains and dyes that are used to identify the
chromosomes.
• Q Banding
o Chromosomes are stained with quinacrine.
• G Banding
o Chromosomes are first digested with
trypsin/pancreatin then stained with Giemsa.
o Stains the regions of DNA that are rich with
adenine and thymine. CLASSIFICATION OF CHROMOSOME
• C Banding • Group A
o Chromosomes are treated with acid and base o Chromosomes 1-3
solutions then stained with Giemsa. o Largest with metacentric chromosomes.
o Stains areas of heterochromatin (tightly • Group B
packed and contains repetitive DNA). o Chromosomes 4-5
• R Banding o Large with submedian centromeres.
o Uses olivomycin or acridine orange then • Group C
stained with Giemsa. o Chromosomes 6-12
o R: Reverse of G Banding. o Medium sized with sub-median centromere.
o Hot salt solution can also be used to treat the • Group D
cell = denatures the DNA. o Chromosomes 13-15.
• NOR-staining o Medium sized with acrocentric centromere.
o NOR: “Nucleolar Organizing Region”.
• Group E
o Silver staining method that identifies genes o Chromosomes 16-18
for ribosomal RNA.
o Short with median or submedian
centromere.
• Group F
o Chromosomes 19-20
o Short with median centromere.
• Group G
o Chromosomes 21-22
o Very short with acrocentric centromere.
• Chromosome X similar to Group C.
• Chromosome Y similar to Group G.
• Majority of the chromosomes in our body is under
Group C.

WRITING NOTATION OF A KARYOTYPES

• Chromosome Analysis or Karyotyping
o Essential tool for studying the genome of an
individual and for the diagnosis of genetic
disorder.
• Karyotype
o Provides us with set of characteristics that
identifies and describes a particular set of
chromosomes such as its relative size,

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CYTOGENETICS
position of centromere, length of
chromosome arms and presence of
secondary contrictions and satellite.
o All of these are translated in the resulting
karyogram.
• Results of karyotype are written in special notations
which can be done as follows:
o In a karyogram, chromosomes appear in
pairs, one from the paternal and the other
from the maternal chromosomes. Count the
number of pairs of chromosomes in the
karyotype. Normally there are 22 pairs of
regular chromosome or autosomes except
the last two in the set which are the sex
chromosomes. Write this number.
§ Referring to the sample karyotype
given, the number will be 46, a
characteristic of a normal human
chromosome set.
o Determine the sex chromosome of the
subject, whether they are “XX” or “XY”. If
they are “XX”, the subject is a female;
“XY”, the subject is a male. Write this
combination next to the number after a
comma. In the given karyotype, the sex
chromosomes that are demonstrated are that
of a female thus the notation will look like
this “46, XX”.
o Note any irregularities in the karyotype. If
the karyotype has an extra chromosome in
autosome number 21, then write “47, XX,
+21, Trisomy-21”, indicating the subject is a
woman with a total of 47 chromosomes and
the extra chromosome is observed in the 21st
pair. Having three chromosomes in a pair is
called “trisomy”. If an extra chromosomes
occurs in the sex chromosome, write 47,
then the sex chromosomes; for example,
“47, XXX”.
MOLECULAR BASIS OF HEREDITY:
DNA, RNA, AND PROTEINS
COMPOSITION AND STRUCTURE OF DNA
• 3 basic components of a DNA molecule:
o Nitrogenous bases
§ Purine bases
• Adenine
• Guanine
§ Pyrimidine bases
• Thymine
• Cytosine
• In RNA: Uracil instead of
Thymine
o Pentose sugar
§ Deoxyribose in DNA
§ In RNA: Ribose
o Phosphate group
CANO
• Nucleoside = pentose sugar + nitrogen base
• Nucleotide = phosphate group + pentose sugar +
nitrogen base
• Watson and Crick in 1953
o Demonstrated how the 3 components are
physically assembled to form DNA.
o Double helix model – DNA is like a twisted
ladder with chemical bonds as its rungs.
o Nucleotides are joined to form a
polynucleotide chain.
o Phosphodiester bonds
§ Link sugar components of adjacent
nucleotides.
o Phosphate attached to the 5’ carbon of one
sugar is linked to hydroxyl group attached to
the 3’ carbon of the next sugar.
o The 5’ – 3’ – 5’ – 3’ orientation of these
linkages continues throughout the chain.
o The asymmetry of the ends of a DNA strand
is the chemical basis of its polarity:
§ One end of the strand is the 5’ end
(terminates in a phosphate).
§ Other end is the 3’ end (terminates
in a hydroxyl).
• Nitrogenous base pairings
o Adenine and Thymine
o Guanine and Cytosine
o In RNA: Adenine and Uracil
• Hydrogen bonds contribute to holding the strands
together.
o A – T pair has two hydrogen bonds
o G – C pair has three hydrogen bonds
• In a DNA duplex, the 5′ end of one strand is opposite
the 3′ end of the other.
o Have opposite orientations = antiparallel.
• Major and minor grooves
o Important for the attachment of DNA
Binding Proteins involved in replication and
transcription.
DANICA YAMBAO 2OMT
CYTOGENETICS
CENTRAL DOGMA OF MOLECULAR BIOLOGY
• Describes the flow of genetic information from DNA
to RNA in order to make a functional product which
is proteins.
• Replication and Transcription occurs in the nucleus.
• Translation occurs in the cytoplasm.
REPLICATION
• Results in the duplication of nuclear DNA in
preparation for cell division.
• Important in duplicating the DNA so that there will
be sister chromatids at anaphase stage and at the end
of cytokinesis, each daughter cell would have the
genetic material.
• Major steps:
o Unwinding of the double stranded DNA
o DNA synthesis
o Rewinding of the double helix
• Proceeds in the 5’-3’ direction.
• Leading Strand
o Oriented in the 3’ to 5’ direction.
o Towards the replication fork.
o Formed continuously.
• Lagging Strand
o Oriented in the 5’ to 3’ direction.
o Away from the replication fork.
o Formed in short sequences of nucleotides =
“Okazaki fragments”.
• Y shape called a replication fork.
• Semiconservative
o Half of chain is part of the original DNA
molecule and half is brand new.
1. Unwinding of parental strands through helicase
protein binding creating a replication fork.
2. Stabilizing the ssDNA through the single-strand
binding proteins at the replication fork.
3. Primase binding at the replication fork which
attracts short complementary RNA, the RNA
primer, at the DNA template.
4. DNA polymerase binding at the DNA template
which adds DNA nucleotide at the RNA primer.
5. Adding of nucleotides by DNA polymerase in a 5’
to 3’ direction to both Parental DNA template.
6. The other strand produced short fragments
(Okazaki fragments), known as discontinuous
synthesis.
7. DNA polymerase proofreading the newly
synthesized DNA and replacing incorrect bases.
8. Annealing helicase rewinding the DNA double
helix and ligase sealing the sugar phosphate.
CANO
TRANSCRIPTION
• Process by which an RNA sequence is formed from a
DNA template.
• Major steps:
o Initiation
o Elongation
o Termination
• Messenger RNA (mRNA)
o Type of RNA produced by the transcription
process.
Initiation:
1. One of the RNA polymerase enzymes (RNA
polymerase II for mRNA) binds to a promoter site
on the DNA.
o Promoter – a nucleotide sequence that lies
just upstream of a gene.
o Promoter sequence determines which of
the two DNA strands will serve as the
template for mRNA synthesis.
2. RNA Pol pulls a portion of the DNA strands apart
from each other, exposing unattached DNA bases.
3. One of the two DNA strands provides the template
for the sequence of mRNA nucleotides.
o Antisense strand – template DNA strand;
3’-5’ direction.
o Sense strand – other DNA strand that
doesn’t serve as template; 5’-3’ direction.
Elongation:
4. The RNA sequence is synthesized only in 5’ to 3’
direction.
o RNA Pol moves in 3’ to 5’ direction
along the DNA template strand while
assembling mRNA strand from 5’ to 3’.
Termination:
5. Transcription continues until a group of bases
called termination sequence is reached.
6. DNA strands and RNA Pol separate from the
transcribed single mRNA strand (primary
transcript).
DANICA YAMBAO 2OMT
CYTOGENETICS
• Post-transcription Modifications
o Modifications that occur in a primary
mRNA molecule before it leaves the
nucleus.
o RNA products of transcription are not
necessarily functional RNAs.
o Processes:
§ Splicing
§ Capping
§ Polyadenylation
o Splicing
§ Introns in the precursor mRNA are
excised, and the exons are spliced
together to form a shorter mature
mRNA.
o Capping
§ 5’ end of RNA is capped by the
addition of a methylated guanine
nucleotide.
o Polyadenylation
§ 3’ end acquires a poly(A) tail that
contains approximately 200
adenine residues.
CANO
TRANSLATION
• Process in which mRNA provides a template for the
synthesis of a protein.
• mRNA cant bind directly into amino acids, instead it
interacts with molecules of tRNA.
• tRNA are cloverleaf shaped RNA stands of about 80
nucleotides.
• Major steps:
o Initiation
o Elongation
o Termination
• Messenger RNA, ribosomal RNA (rRNA) and
transfer RNA (tRNA) all participate in translation.
• Ribosomes – site of protein synthesis.
o Made up of two different sized subunits,
which consist of different types of rRNA
molecules and many ribosomal specific
proteins.
Initiation:
1. The ribosome finds the initiation site on the
mRNA sequence.
o Initiation site = AUG codon (start codon)
which specifies for Methionine (usually
removed from the polypeptide before the
completion of the synthesis).
Elongation:
2. The ribosome binds the tRNA to its surface so that
base pairing can occur between tRNA and mRNA.
3. The ribosome moves along the mRNA, codon by
codon, in the 5’ to 3’ direction.
4. As each codon is processed, an amino acid is
translated by the interaction of mRNA and tRNA.
5. The ribosome provides an enzyme that catalyzes
the covalent peptide bonds between adjacent
amino acids, resulting in a growing polypeptide.
Termination:
6. Termination of translation occurs when the
ribosome arrives at a stop codon on the mRNA
sequence.
o Stop codon: amber (UAG), opal/umber
(UGA), and ochre (UAA)
7. Upon completion of synthesis, the mRNA,
ribosome and polypeptide separate from one
another.
8. The polypeptide is released into the cytoplasm.
DANICA YAMBAO 2OMT
CYTOGENETICS CANO
*Other types of RNAs have a regulatory role or are involved
in processing pre-RNAs.

THE GENETIC CODE

• Total of 64 codons: 3 stop codons and 61 codons that
code for 20 amino acids (where one codon is the start
codon [AUG→Met])

• Post-translation Modifications
o Modifications that occur in newly
synthesized polypeptides to become
functional proteins.
o Examples:
§ Proteolytic cleavage into smaller
polypeptide units.
§ Combination with other
polypeptides to form larger
proteins.
§ Addition of carbohydrate or lipid
moieties.
§ Modification of amino-acid side
chains.
o Osteogenesis imperfecta
§ Disease caused by defects in the
formation of bone.
§ “Brittle bone disease”.
§ Post-translational modification in
type I collagen.
MAIN TYPES OF RNA
GENETIC MUTATIONS
• A mutation is any heritable change in the amount or
structure of genetic material.
• Classification of mutations can be based on:
o Origin
o Cell type
o Expression
o Effect on function
o Molecular change and its effects on protein
products
• Mendelian disorders, complex multigenic disorders
and chromosomal disorders are not included.
BASED ON ORIGIN
• Spontaneous
o Occurs in absence of known mutagen (an
agent that changes genetic material).
o Statistically random, unpredictable events.
• Induced
o Occurs in presence of known mutagen.
• Each gene has its rate of mutation.
o Can increase by treatment with a mutagen or
radiation.

BASED ON CELL TYPE

• Somatic

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CYTOGENETICS CANO
o Occurs in nonreproductive cells (not o Qualitatively alters the action of a gene.
passable). § Example: cause a gene to become
o Can yield a genotypic mixture (mosaic) of active in a type of cell or tissue in
normal and mutant tissue. which the gene is not normally
o Most common cancers. active.
o Can simply contribute to genetic diversity.
• Germ line BASED ON MOLECULAR CHANGE
o Occurs in reproductive cells. • In molecular genetics, base-pair substitutions are also
o Inherited. termed point mutations.
o May be silent, may cause a disease, or • In classical genetics, point mutations denote any
generate beneficial genetic diversity. mutation small enough to be unobservable under a
microscope.
BASED ON EXPRESSION • Substitutions
• Conditional o Most common type of mutation.
o Expressed only under restrictive conditions. o Replacement of a single nucleotide by
§ Example: high temperature another.
o Effect of mutation can be turned on or off by o Transition
experimenter. § Replacement by the same type of
§ Useful for genetic analysis. nucleotide pyrimidine for a
• Unconditional pyrimidine (C for T or vice versa).
o Expressed under permissive conditions as § Purine for a purine (A for G or vice
well as restrictive conditions. versa).
• Example: a temperature-sensitive mutation can cause § Most frequent due to CpG
cell death at high temperature (restrictive condition), dinucleotides.
but might have no deleterious consequences at a § CpG dinucleotides
lower temperature (permissive condition). • Likely to be the result of
the nucleotides cytosine
BASED ON EFFECT ON FUNCTION and guanine occurring
• Loss of function (knockout or null mutation) together frequently being
o Creates a null allele. methylated in genomic
o Functionally equal to a deletion of a gene. DNA with spontaneous
o Eliminates normal function. deamination of
o Results in complete gene inactivation or in a methylcytosine converting
completely nonfunctional gene product. them to thymine.
§ Deletion of all or part of a gene. • P represents the
§ Amino acid replacement that phosphate.
inactivates the protein. • Termed “hotspots” for
• Hypomorphic (leaky mutation) mutation.
o There is some expression. o Transversion
o Reduces normal function but does not § Substitution of a pyrimidine by a
eliminate the level of expression of a gene or purine or vice versa.
the activity of the gene product.
§ Individuals may have enough
enzyme activity to “leak through”
to produce a quasi-normal
phenotype.
• Hypermorphic
o Over expression or over activity. • Deletions
o Increases normal function. o Involves the loss of one or more nucleotides.
o Produces a greater-than-normal level of gene • Deletion patterns:
expression. o Multiple of 3 nucleotides (codon)
§ Mutation changes the regulation of § Deletion of amino acids that may
the gene so that the gene product is affect protein function or stability/
overproduced. o Not multiple of 3
• Gain of function (ectopic expression) § Likely results to frameshift
o Ecto = out of place. mutation.
o Gene is not usually expressed. o Large deletion
o Expressed at incorrect time or in
inappropriate cell types.

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CYTOGENETICS CANO
§ May arise through unequal • Caused by length of a
crossover between repeat repeated section of a gene
sequences. exceeding a normal range.
§ Either: partial gene deletion or • HTTG is located on the
whole gene deletion short arm of chromosome
• Three-base deletion in the common cystic fibrosis 4.
(CF) allele results in synthesis of a protein that lacks • CAG codes for glutamine.
amino acid 508 (phenylalanine). • Chain of glutamine =
polyglutamine tract.
• Repeated part of the gene
= polyQ region.
§ Fragile X syndrome (FMR1 gene)
• CGG repeats.
• Repeats in the 5’
untranslated region of
MR1 gene.
• Insertions § Myotonic dystrophy (DMPK gene)
o Involves the addition of one or more • CTG repeats.
nucleotides into a gene. • DMPK gene/ Myotonic
• Insertion patterns: dystrophy protein kinase:
o Multiple of 3 nucleotides (codon) predominantly expressed
§ Insertion of amino acids that may in skeletal muscles;
affect protein function or stability. located on long arm of
o Not multiple of 3 chromosome 19.
§ Likely results to frameshift § Friedreich ataxia (FXN1 gene)
mutation. • GAA repeats.
o Large insertion • Autosomal recessive
§ Results from unequal crossover disorder that affects a gene
(e.g. hereditary sensory and motor on chromosome 9.
neuropathy type 1a) or the insertion • Produces frataxin
of transposable elements. (protein).
§ Either: partial gene duplication or
whole gene duplication EFFECTS OF MUTATIONS ON PROTEIN PRODUCTS
o Expansion of trinucleotide repeat (most • Divisions
contain C and/or G) o Synonymous Mutations
§ Amplification of a sequence of § Silent mutations
three nucleotides which prevents o Nonsynonymous Mutations
normal expression of the gene. § Missense mutations
§ Involves dynamic mutations § Nonsense mutations
wherein the repeat sequence § Frameshift mutations
becomes more unstable as it • Synonymous Mutations
expands in size. o Point mutations = miscopied DNA
§ The exact mechanism are not nucleotide that only changes one base pair in
completely understood. the RNA copy of the DNA.
§ Unstable trinucleotides repeats may o Mutation does not alter the polypeptide
be within coding or noncoding product of the gene.
regions of genes and hence vary in o Silent mutations
their pathogenic mechanisms. § Base substitution occurs but does
§ Possible causes: not change the amino acid
• Unequal sister chromatid sequence.
exchange on
nonreplicating DNA.
• Slipped-strand mispairing
and polymerase slippage
in replicating DNA.
§ Huntington disease (HTT gene)
• CAG repeats.

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CYTOGENETICS CANO
• Nonsynonymous Mutations
o Much greater effect. § β0-thalassemia
o Insertion or deletion of a single nucleotide • Affects the β-globin chain
sequence during transcription when mRNA of hemoglobin.
is copying the DNA. • The codon for glutamine
o Mutation leads to an alteration in the (CAG) creates a stop
encoded polypeptide. codon (UAG) as U is
o Likely to result in abnormal function, which substituted for C.
is usually associated with disease or
lethality.
o Missense mutations
§ Arise from point mutations.
§ Base-pair substitutions that produce
a change in a single amino acid.
§ Change in amino acid may affect
the protein structure.

o Frameshift mutations
§ Sickle cell anemia
• Affects the β-globin chain
of hemoglobin.
• Substitution of glutamic
acid to valine.
§ Framing error or reading frame
shift.
§ When a mutation involves the
insertion or deletion of nucleotides
that are not a multiple of three, it
will disrupt the reading frame.
§ Also alter the first stop codon.
§ Polypeptide could be abnormally
short or abnormally long and will
not be most likely functional.
§ Resulting amino acid sequence
bears no resemblance to the normal
sequence and may have an adverse
effect on its function.

o Nonsense mutations
§ Arise when a mutation in a DNA
sequence causes a protein to
terminate prematurely by changing
the original amino acid to a stop § Single-base deletion at the ABO
codon. (glycosyltransferase) locus, leading
§ Base-pair substitutions that produce to a frameshift mutation
a stop codon in the mRNA. responsible for the O allele.
§ Result in a premature termination
of the polypeptide chain.
§ Unlikely to retain normal biological
activity, especially when there is a
loss of an important functional
domain(s) of the protein.

DANICA YAMBAO 2OMT

CYTOGENETICS
§ Tay-Sachs disease: four-base
insertion in the hexosaminidase A
gene
*Mutations can range from single base substitutions, through
insertions and deletions of single or multiple bases to loss or
gain of entire chromosomes.
*Base substitutions are most prevalent and missense mutations
account for nearly half of all mutations.
ROLE OF CANCER GENES IN GENETIC MUTATIONS
BASIC GENETICS OF CANCER
• All cancer is a genetic disease of somatic cells
because of aberrant cell division or loss of normal
programmed cell death.
• A small proportion is strongly predisposed by
inherited germline mutations behaving as Mendelian
traits.
• For many cancers, environmental factors are
etiologically important, whereas heredity plays a
lesser role.
MAJOR CLASSES OF CANCER GENES
• Oncogenes
• Tumor suppressor genes
• DNA repair genes
ONCOGENES
• Affect cell growth or development that can cause
cancer.
• Genes that induce a transformed phenotype when
expressed in cells by promoting increased cell
growth.
• Cancer genes.
• Proto-oncogenes
o Involved in four basic regulators of normal
cell growth:
§ Growth factors
CANO
§ Growth factor receptors
§ Signal transduction molecules
§ Nuclear transcription factors
o Normal cellular genes that can become
mutated and overexpressed to become
oncogenes.
o If gene undergoes translocation (next to
highly expressed gene), it will lead to gain-
of-function mutations and become
oncogenes.
• Growth factor genes
o Stimulate cells to grow by binding to growth
factor receptors and they govern the
transition of a cell from G0 to the start of the
cell cycle.
o Example: v-SIS oncogene
§ Normally encodes part of the
biologically active platelet-derived
growth factor subunit.
§ When added to cell cultures, they
are transformed, behaving like
neoplastic cells – their growth rate
increases, and they lose contact
inhibition.
o Example: ERBB2/HER2/NEU gene
§ Codes for protein that is receptor
for epidermal growth factor.
§ Gene duplication will lead to
signaling the cell to activate
transcription of genes whose
protein products stimulate cell
division and eventually causing
breast cancer.
§ HER2/NEU
• Approximately amplified
in 20-30% of breast
carcinomas.
• Protein product: growth
factor receptor located on
the surfaces of breast
cancer cells.
• Growth factor receptors genes
o Encode proteins that form growth factor
receptors, possessing tyrosine kinase
domains that allow cells to bypass normal
control mechanisms.
o Example: KIT gene
§ Mutations in KIT occur in sporadic
and hereditary gastrointestinal
stromal tumor syndrome (known as
GIST) as a result of point
mutations.
• Signal transduction genes
o Forms of signal transduction: proteins with
GTPase activity and cytoplasmic serine
threonine kinases.
o Example: RAS genes
§ Mutations lead to increased or
sustained GTPase activity.
§ Excessive cell division.
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CYTOGENETICS CANO
o Example: BRAF genes insensitivity to anti-growth signals and
§ Mutations result in sustained or evasion of apoptosis.
increased transmission of a growth- § Eliminates the DNA checkpoint
promoting signal to the nucleus. that monitors DNA damage in G1
§ Malignant melanoma and colon and S.
cancer. o Nonfunctional in more than half of all
• Transcription factor genes cancers.
o May activate or suppress neighboring DNA • E-cadherin
sequences. o Ubiquitously expressed on epithelial cells.
o Example: NMYC gene o Acts to suppress invasion and metastasis by
§ DNA-binding protein. epithelial cells.
§ Mutations activate expression of o Inactivation is a key step in acquisition of
many genes through binding the capability for metastasis.
enhancer box sequences and o Loss of function has been implicated in
recruiting histone cancer progression and metastasis.
acetyltransferases. • RB gene
§ Mutations occur in neuroblastoma o First tumor suppressor protein discovered in
and lung cancer. human retinoblastoma.
o Functions:
§ Gatekeeper that blocks cell
proliferation.
§ Regulate cell division.
o Retinoblastoma protein controls the
transition from G1 to S phase by controlling
the activity of the transcription factor E2F.
o Loss of RB function frees E2F to initiate
transcription of the enzymes for DNA
synthesis at all times in the cell cycle.
§ Excessive rounds of DNA synthesis
are continuously being initiated.
• WT1 gene
o Associated with embryonic malignancy of
the kidney.
TUMOR SUPPRESOR GENES o Normally halts mitosis in the rapidly
developing kidney tubules in the fetus.
• Normally control cell proliferation and activate the
o If deleted, the congenital anomaly would
apoptotic pathway.
retain pockets of actively dividing cells that
• Genes that normally prevent uncontrolled growth. would result to tumor called Wilm’s tumor.
• If gene undergoes deletion, it will lead to loss-of-
function mutations which may induce uncontrolled
mitosis.
• Inherited mutations are dominant alleles at the level
of the individual (i.e., heterozygotes usually develop
the disease), but they are recessive alleles at the level
of the cell (heterozygous cells do not form tumors).
o Resolved by realizing that in individuals
who have inherited the first hit, a second hit
that occurs in any one cell will cause a
tumor.
• p53 gene
o
o Caretake gene.
o Functions:
§ DNA repair DNA REPAIR GENES
§ Induce apoptosis • Resolve mistakes made when DNA is copied.
§ Transcription • If a person has an error in a DNA repair gene,
§ Regulate cell cycle mistakes remain uncorrected and these become
o Loss of function of p53 results in acquisition mutations
of two characteristics of cancer cells: • Defects in DNA repair result in greatly elevated
mutation rates and genetic instability.

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CYTOGENETICS
• Mutant cells have higher mutation rates, which
promote progression toward the cancerous state.
• Defects in any gene that encode proteins involved in
DNA mismatch repair cause hereditary nonpolyposis
colorectal cancer (HNPCC), which shows autosomal
dominant inheritance.
• Inherited breast cancer syndromes prove to be
associated with mutations in either of two genes,
BRCA1 or BRCA2, which are involved in repair of
double-strand breaks.
• Xeroderma pigmentosum cells (skin cancer) are
defective in nucleotide excision repair; they are
unable to repair defects such as thymine dimers that
are induced by ultraviolet light.
FLUORESCENSE IN-SITU HYBRIDIZATION (FISH)
• A cytogenetic technique that uses fluorescent probes
that bind specifically to a part of chromosomes
complimentary to its sequence.
• Useful in detecting and mapping the presence or
absence of particular DNA sequences within
chromosomes.
SPECIMEN TYPES FOR FISH
*Fluorescently labeled DNA probes can be hybridized to cells
in both interphase and metaphase stages of the cell cycle.
*FISH can be applied to a variety of specimen types
depending upon the study of interest.
CANO
FISH PROBES
• Complementary sequences of target nucleic acids
(DNA, RNA or nucleic acid analogs) tagged or
labeled with fluorophores.
• Size ranges from 20 to 1000 base pairs.
• Direct Labeling
o Fluorophores are directly attached to the
probe.
o Less sensitive.
o Example: FITC, rhodamine, and cyanines
• Indirect Labeling
o Chemical conjugation of the nucleic acid
with a nonfluorescent molecule that can bind
fluorescent material after hybridization.
o Probes can be indirectly labeled via
incorporation of a hapten (such as biotin or
digoxigenin) into the DNA via nick
translation or PCR.
o Haptens
§ Attached to the probe nucleotides.
§ Detected by a secondary reaction
using a fluorescently labeled
antibody.
o While indirect labeling has the potential for
generating greater fluorescence signal, it
also has the disadvantage of requiring
additional incubation steps to bind the
antibody and avidin reagents.
o The introduction of fluorescent antibodies
also can increase the background
fluorescence owing to nonspecific binding
of the antibodies and avidin proteins to
extraneous cellular material on the
microscope slide, and the slide surface itself.
TYPES FISH PROBES
• Locus Specific Probe
• Alphoid or Centromeric Repeat Probe
• Subtelomeric Probe
• Whole Chromosome Probe
• Pre-natal FISH Probe
LOCUS SPECIFIC PROBE
• Binds to a particular region of a chromosome.
• Used when only a small portion of a gene is isolated
and want to determine on which chromosome the
gene is located, or how many copies of a gene exist
within a particular genome.
DANICA YAMBAO 2OMT
CYTOGENETICS CANO
ALPHOID OR CENTROMERIC REPEAT PROBE
• Generated from repetitive sequences found in the
middle of each chromosome.
• Used to determine whether an individual has the
correct number of chromosomes or if there is
aneuploidy in the patient’s genome.

SUBTELOMERIC PROBE
• Specific to the subtelomere region of chromosome.
• Useful in the detection of subtelomere deletions and
rearrangements.

WHOLE CHROMOSOME PROBE

• Collection of smaller probes that bind to the whole
length of chromosome.
• Useful in the examination of chromosomal
aberrations.

PRE-NATAL FISH PROBE

• Comprise of different combinations of fluorophore-
labeled probes specific for chromosomes 13, 18, 21,
X, and/or Y.

STEPS
1. Probe and target DNAs are denatured using high
temperature incubation in a formamide/
salt solution.
2. Probe sequences hybridize to the complementary • Two color (e.g., X and Y) counting guidelines
target sequences, and nonspecific binding is
eliminated via stringent washing.
3. The probe hybridization is detected with fluorescence
microscopy.

CELL SCORING IN FISH

• FISH signal patterns may be scored manually by
qualified technologists, or computerized automated
“spot” counting maybe incorporated into the analysis.
• Single color FISH counting guidelines

DANICA YAMBAO 2OMT

CYTOGENETICS
Example:
• Cells are from an uncultured amniocyte preparation
that was hybridized with a locus specific probe for 13
(green) and 21 (red).
• Upper cell: 2 signals for both probes
• Lower cell: 3 signals for chromosome 21 and 2
signals for chromosome 13
APPLICATION OF FISH
• Detection and characterization of chromosome
abnormalities
o Example of FISH to a single-copy target
using a cosmid (SNRPN) to the Prader-Willi
“critical region” localized to 15q11-13.
o (a) A metaphase in which one normal
chromosome 15 has three hybridization
signals from a centromeric control probe
(green), a distal control probe (red), and a
probe to the critical region (red). The other
chromosome 15 (arrow) revealed
hybridization signals only for the two
control probes. Thus, this chromosome was
deleted for the critical region, and this
patient was diagnosed with Prader-Willi
syndrome. Chromosomes were
counterstained blue with DAPI.
o (b) In this partial metaphase, a SNRPN
probe and a control probe (both yellow)
were utilized (current standards and
guidelines require the use of different
fluorochromes). Chromosomes were
CANO
counterstained orange with propidium
iodide. The arrow indicates the chromosome
15 with a duplicated SNRPN signal.
o Partial metaphase spread from a patient with
a duplication involving chromosome 11
o A BAC localized to chromosome 11p15.5
produced one signal on the normal
chromosome 11 and a double signal on the
duplicated chromosome 11 (arrow). The
duplication in the short arm of chromosome
11 was detected in a newborn that was large
for gestational age. The infant also had an
omphalocele and was diagnosed with
Beckwith-Wiedemann syndrome.
• Detection and analysis of prenatal chromosomal
abnormalities
o Prenatal ploidy assessment utilizing Abbott
Molecular AneuVysion TM analysis of
uncultured amniotic fl uid cells using unique
copy probes for the long arms of
chromosomes 13, 18, 21, X, and Y.
o The results in these interphase cells are
consistent with a XY fetus with trisomy 21.
o Left: probes for chromosomes 13 (2 green
signals) and 21 (3 orange signals).
o Right: probes for chromosomes 18 (2 aqua
signals), X ( green signal), and Y (orange
signal).
o Nuclei are counterstained blue with DAPI.
• Study of chromosomal abnormalities associated with
cancer
o FISH panels for B cell disorders. Results
from a peripheral blood sample from a
DANICA YAMBAO 2OMT
CYTOGENETICS
patient with CLL, hybridized with the
Abbott Molecular CLL probe panel with
addition of an MYB probe.
o Left: shows a deletion of 13q14 (single red
signal and two aqua signals for the locus on
13q34) and a normal signal pattern for the
chromosome 12 centromere probe.
o Right: shows deletion of TP53 (single red
signal), deletion of ATM (single green
signal), and a normal signal pattern for the
MYB probe at 6q23.
o ERBB2 (HER2) analysis for carcinoma of
the breast.
o Green signals represent the chromosome 17
centromere probe, while the ERBB2 probe
signals are red. An ERBB2:17 centromere
ratio of ³ 2.0 represents amplification of the
ERBB2 gene.
o (a) Normal cells, with two red and two green
signals.
o (b) ERBB2 amplification.
SPECIALIZED AND EVOLVING TECHNIQUES
COMPARATIVE GENOMIC HYBRIDIZATION (CGH)
ON METAPHASE CELLS
• A technique that uses DNA from the cells of interest,
rather than using a standard karyotype, for
chromosomal analysis.
• This can be very useful, especially in some cancers
when only DNA is available rather than any growing
cells.
• This technology has been used successfully for
clinical analysis, particularly with cases that have a
low (or no) mitotic index.
CANO
• It is not useful for detecting balanced rearrangements.
• The utility of metaphase CGH is illustrated by the
CGH profiles of a case with an insertion of unknown
material into the short arm of chromosome 4. The
chromosomal profiles reveal a gain of 15q
(highlighted in orange).
MULTIPLEX FISH (M-FISH)
• A technique that allows the investigator to view a
karyotype so that each chromosome is “painted” with
a different color.
• Ratio-labeled probes are used to create a distinct
computer-generated false color for each chromosome.
• Useful for complex rearrangements, such as those
seen in neoplastic disorders and solid tumors.
• M-FISH of the pre-B ALL derived cell line RS4; 11
showing (A) blended colours generated by merging
the separate fluorochrome images, (B) pseudocolours
generated using the colour scheme in Fig. 1, and (C)
colour karyotype showing the t(4; 11)(q21;q23), i(7q)
and trisomy 8.
MULTICOLOR BANDING (MBAND) ANALYSIS
• Uses chromosome-specific mixtures of partial
chromosome paints that are labeled with various
fluorochromes.
• A computer program analyzes metaphase
chromosome data and produces a pseudocolored,
banded karyotype with an estimated resolution of 550
bands, regardless of chromosome length.
DANICA YAMBAO 2OMT
CYTOGENETICS CANO
• Advantageous for the determination of breakpoints
and the analysis of intrachromosomal rearrangements
and can be particularly useful in preparations with
shorter chromosomes.
• Multicolor banding. (a) Region-specific probes
labeled with different partial chromosome paints
(PCP) and computer false color (MetaSystems’
mBAND) produces a definable number of colored
bands per chromosome, regardless of chromosome
length. (b) This example shows an abnormal X
chromosome (right homolog of each pair).

PRIMED IN SITU LABELING (PRINS)

FIBER FISH
• A technique that is almost entirely used for research.
• It allows the chromosomes to be stretched out and
elongated.
• The probes are applied and can be physically ordered
on the fibers.
• This provides a much higher spatial resolution and
allows for correct orientation and placement of
probes and for precise mapping of probes.
• Fiber-FISH analyzing the association of CL14 and
CL34 repeats with telomeric DNA. (a) Five
representative fiber-FISH signals derived from
probes pAtT4 (red) and CL14 (green). (b) Five
representative fiber-FISH signals derived from
probes pAtT4 (red) and CL34 (green). Only the most
proximal part of each CL14/CL34 signal was
included in each image. Note the different sizes of the
telomeric DNA and gap in the junctions, indicating
these signals may be derived from different
chromosomal ends. (c) Fiber-FISH analysis of
telomere (yellow), CL34 (red), and CL14 (green).
The CL34 signals within the four images show
significantly different sizes. Only the most proximal
part of each CL14 signal was included in the image.
• Essentially PCR on a slide.
• Primers of interest are hybridized on a slide and then
subjected to cycles of denaturation, reannealing, and
elongation that are used to incorporate labeled
nucleotides. The labels are then detected
fluorescently, or labeled nucleotides are incorporated
during the reaction.
• Can differentiate hybridization with the alpha satellite
sequences for chromosomes 13 and 21, something
that cannot be done with traditional FISH.
• Metaphase chromosomes are subjected to PRINS
with alpha satellite oligonucleotides specific for
chromosomes X, 11, and 17. Bright yellow
fluorescein staining is seen at the centromeres of
these chromosomes.
REVERSE FISH
• Used to identify material of unknown origin.
• This unidentified material, such as a marker
chromosome or duplication, is flow sorted or
microdissected off of a slide after G-banding. The
DNA from this material is extracted, PCR- amplified
and labeled with a fluorochrome. This is then used as
a probe and hybridized to normal or patient
metaphase chromosomes to identify the origin of the
unknown material.
• Reverse FISH of a patient with an abnormal
chromosome 8 (a) G- banding suggested a
duplication of bands 8p23.1 ® p23.3. Two pairs of
chromosomes 8 are shown; arrows indicate the
additional band. This band was microdissected, and
the DNA was amplified, labeled, and used as a FISH
DANICA YAMBAO 2OMT
CYTOGENETICS CANO
probe (b,c) Hybridization to normal chromosomes.
(b) The same metaphase is imaged with reverse DAPI
to approximate G-banding patterns and identify the
two chromosomes 8, and with (c) typical DAPI
staining. Arrows indicate both chromosomes 8 (d)
Hybridization back to a metaphase from the patient,
demonstrating that one chromosome 8 contains a
duplication (arrow). The reverse FISH results
confirm the initial interpretation.

DANICA YAMBAO 2OMT