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ABSTRACT
During the Metaphase type of cell division in the Bone Marrow,
Karyotyping method gives the visual representation of the 46 chromosomes paired
and arranged in decreasing order of size. This representation is useful in leukemia
purposes. This method is a difficult one because these chromosomes appear
distorted, overlapped, and their images are usually blurred with undefined edges.
So here, Karyotyping uses new mutual information method which is proposed to
increase the discriminate power of the G-banding pattern dissimilarity between
chromosomes and improve the performance of the classifier. This algorithm is
formulated as such a method of combinatorial optimization problem. Where the
distances between homologous chromosomes are minimized and the distances
between non homologous ones are maximized. It is solved by using an integer
programming approach. In this project chromosome dataset Lisbon-K1 (LK1)
chromosome dataset with 9200 chromosomes was used for this study.
CHAPTER 1
Introduction 1.1
The study of chromosome morphology and its relation with some genetic
diseases is the main goal of cytogenetic. Normal human cells have 23 classes of
large linear nuclear chromosomes, in a total of 46 chromosomes per cell. The
chromosome contains approximately 30 000 genes (genotype) and large tracts of
non coding sequences. The analysis of genetic material can involve the
examination of specific chromosomal regions using DNA probes, e.g., fluorescent
in situ hybridization (FISH) called molecular cytogenetic, comparative Genomic
hybridization (CGH) , or the morphological and pattern analysis of entire
chromosomes, the conventional cytogenetic, which is the focus of this paper.
These cytogenetic studies are very important in the detection of acquired
chromosomal abnormalities, such as translocations, duplications, inversions,
deletions, monosomies, or trisomies.
These techniques are particularly useful in the diagnosis of cancerous
diseases and are the preferred ones in the characterization of the different types of
leukemia, which is the motivation of this paper . The pairing of chromosomes is
one of the main steps in conventional cytogenetic analysis where a correctly
ordered karyogram is produced for diagnosis of genetic diseases based on the
patient karyotype. The karyogram is an image representation of the stained human
chromosomes with the widely used Giemsa Stain metaphase spread (G-banding) ,
where the chromosomes are arranged in 22 pairs of somatic homologous elements
plus two sex-determinative chromosomes (XX for the female or XY for the male),
displayed in decreasing order of size. A karyotype is the set of characteristics
extracted from the karyogram that may be used to detect chromosomal
abnormalities. The metaphase is the step of the cellular division process where the
chromosomes are in their most condensed state. This is the most appropriated
moment to its visualization and abnormality recognition because the chromosomes
appear well defined and clear.
The pairing and karyotyping procedure, usually done manually by visual
inspection, is time consuming and technically demanding. The application of the
G-banding procedure to the chromosomes generates a distinct transverse banding
pattern characteristic for each class, which is the most important feature for
chromosome classification and pairing. The International System for Cytogenetic
Nomenclature (ISCN) provides standard diagrams/ideograms of band profiles, as
for all the chromosomes of a normal human, and the clinical staff is trained to pair
and interpret each specific karyogram according to the ISCN information. Other
features, related to the chromosome dimensions and shape, are also used to
increase the discriminative power of the manual or automatic classifiers.
1.2 CHROMOSOME
A chromosome is an organized structure of DNA and protein found
in cells. It is a single piece of coiled DNA containing many genes,regulatory
elements and other nucleotide sequences. Chromosomes also contain DNA-bound
proteins, which serve to package the DNA and control its functions.
Chromosomes vary widely between different organisms. The DNA molecule may
be circular or linear, and can be composed of 100,000 to
10,000,000,000[1] nucleotides in a long chain. Typically, eukaryotic cells (cells
with nuclei) have large linear chromosomes andprokaryotic cells (cells without
defined nuclei) have smaller circular chromosomes, although there are many
exceptions to this rule. Also, cells may contain more than one type of chromosome;
for example, mitochondria in most eukaryotes and chloroplasts in plants have their
own small chromosomes.
In eukaryotes, nuclear chromosomes are packaged by proteins into a condensed
structure called chromatin. This allows the very long DNA molecules to fit into
the cell nucleus. The structure of chromosomes and chromatin varies through
the cell cycle. Chromosomes are the essential unit for cellular division and must be
replicated, divided, and passed successfully to their daughter cells so as to ensure
the genetic diversity and survival of their progeny. Chromosomes may exist as
either duplicated or unduplicated. Unduplicated chromosomes are single linear
strands, whereas duplicated chromosomes contain two identical copies
(called chromatids) joined by a centromere.
Compaction of the duplicated chromosomes
during mitosis and meiosis results in the classic four-arm structure (pictured to the
right). Chromosomal recombination plays a vital role in genetic diversity. If these
structures are manipulated incorrectly, through processes known as chromosomal
instability and translocation, the cell may undergo mitotic catastrophe and die, or it
may unexpectedly evadeapoptosis leading to the progression of cancer.
In practice "chromosome" is a rather loosely defined term. In
prokaryotes and viruses, the term genophore is more appropriate when no
chromatin is present. However, a large body of work uses the term chromosome
regardless of chromatin content. In prokaryotes, DNA is usually arranged as a
circle, which is tightly coiled in on itself, sometimes accompanied by one or more
smaller, circular DNA molecules called plasmids. These small circular genomes
are also found in mitochondria and chloroplasts, reflecting their bacterial origins.
The simplest gonophores are found in viruses: these DNA or RNA molecules are
short linear or circular gonophores that often lack structural proteins.
1.3 MUTATIONS IN CHROMOSOME NUMBER
Normally, members of the same species have the same numbers of
types of chromosomes (with the exception of sex chromosomes in males and
females if sex is chromosomally determined). Such individuals are called euploid
and have the wild-type chromosome complement for the species. Euploid human
karyotypes are 46, XX (female) or 46 XY (male).
Chromosomal Mutations are substantial changes in
chromosome structure that are large enough to be visible by karyotyping (see lab
manual) and thus typically affect more than one gene. If the mutation involves
only one or a few chromosomes in the genome (e.g. a extra copy of human
chromosome 21), the individual carrying the mutation is said to be aneuploid. An
example of aneuploidy is trisomy 21, in which an individual has 3, rather than 2,
copies of chromosome 21. The individual would have Down Syndrome and
his/her karyotype would be written 47,+21,XY or 47,+21,XX.
Fig 1.1 chromosome paired model
Aneuploidy is usually caused by spindle fiber failure in meiosis I or II. Such a
failure of the separation of homologous chromosomes or sister chromatids is called
nondisjunction.
1.4 Metaphase chromatin and division
In the early stages of mitosis or meiosis (cell division), the
chromatin strands become more and more condensed. They cease to function as
accessible genetic material (transcription stops) and become a compact
transportable form. This compact form makes the individual chromosomes visible,
and they form the classic four arm structure, a pair of sister chromatids attached to
each other at the centromere. The shorter arms are called p arms (from
the French petit, small) and the longer arms are called q arms (q follows p in the
Latin alphabet; q-g "grande"). This is the only natural context in which individual
chromosomes are visible with an optical microscope.
During mitosis, microtubules grow from centrosomes located at
opposite ends of the cell and also attach to the centromere at specialized structures
called kinetochores, one of which is present on each sister chromatid. A special
DNA base sequence in the region of the kinetochores provides, along with special
proteins, longer-lasting attachment in this region. The microtubules then pull the
chromatids apart toward the centrosomes, so that each daughter cell inherits one set
of chromatids. Once the cells have divided, the chromatids are uncoiled and DNA
can again be transcribed. In spite of their appearance, chromosomes are structurally
highly condensed, which enables these giant DNA structures to be contained
within a cell nucleus.
1.5 BONE MARROW
Bone marrow (Latin: medulla ossium) is the flexible tissue found
in the interior of bones. In humans, bone marrow in large bones produces
new blood cells. On average, bone marrow constitutes 4% of the total body mass
of humans; in adults weighing 65 kg (143 lbs), bone marrow accounts for
approximately 2.6 kg (5.7 lbs). The hematopoietic compartment of bone marrow
produces approximately 500 billion blood cells per day, which use the bone
marrow vasculature as a conduit to the body's systemic circulation.[1] Bone marrow
is also a key component of the lymphatic system, producing the lymphocytes that
support the body's immune system
CHAPTER 2
2.1 MITOSIS
Mitosis is the process by which a eukaryotic cell separates the chromosomes
in its cell nucleus into two identical sets, in two separate nuclei. It is generally
followed immediately by cytokinesis, which divides the nuclei, cytoplasm,
organelles and cell membrane into two cells containing roughly equal shares of
these cellular components. Mitosis and cytokinesis together define the mitotic (M)
phase of the cell cycle—the division of the mother cell into two daughter cells,
genetically identical to each other and to their parent cell. This accounts for
approximately 10% of the cell cycle.
Mitosis occurs only in eukaryotic cells and the process varies in different
species. For example, animals undergo an "open" mitosis, where the nuclear
envelope breaks down before the chromosomes separate, while fungi such as
Aspergillus nidulans and Saccharomyces cerevisiae (yeast) undergo a "closed"
mitosis, where chromosomes divide within an intact cell nucleus.[1] Prokaryotic
cells, which lack a nucleus, divide by a process called binary fission.
The process of mitosis is fast and highly complex. The sequence of events is
divided into stages corresponding to the completion of one set of activities and the
start of the next. These stages are interphase, prophase, prometaphase, metaphase,
anaphase and telophase. During mitosis the pairs of chromatids condense and
attach to fibers that pull the sister chromatids to opposite sides of the cell. The cell
then divides in cytokinesis, to produce two identical daughter cells which are still
diploid cells.
Because cytokinesis usually occurs in conjunction with mitosis, "mitosis" is
often used interchangeably with "mitotic phase". However, there are many cells
where mitosis and cytokinesis occur separately, forming single cells with multiple
nuclei. This occurs most notably among the fungi and slime moulds, but is found
in various different groups. Even in animals, cytokinesis and mitosis may occur
independently, for instance during certain stages of fruit fly embryonic
development. Errors in mitosis can either kill a cell through apoptosis or cause
mutations that may lead to cancer.
Fig 3 Mitosis cell division
The primary result of mitosis is the transferring of the parent cell's genome
into two daughter cells. These two cells are identical and do not differ in any way
from the original parent cell. The genome is composed of a number of
chromosomes—complexes of tightly-coiled DNA that contain genetic information
vital for proper cell function. Because each resultant daughter cell should be
genetically identical to the parent cell, the parent cell must make a copy of each
chromosome before mitosis. This occurs during the S phase of interphase, the
period that precedes the mitotic phase in the cell cycle where preparation for
mitosis occurs.
Each chromosome now has an identical copy of itself, and together the two
are called sister chromatids. The sister chromatids are held together by a
specialized region of the chromosome known as the centromere.
In most eukaryotes, the nuclear envelope which segregates the DNA from
the cytoplasm disassembles. The chromosomes align themselves in a line spanning
the cell. Microtubules — essentially miniature strings— splay out from opposite
ends of the cell and shorten, pulling apart the sister chromatids of each
chromosome. As a matter of convention, each sister chromatid is now considered a
chromosome, so they are renamed to sister chromosomes. As the cell elongates,
corresponding sister chromosomes are pulled toward opposite ends. A new nuclear
envelope forms around the separated sister chromosomes.
As mitosis completes,the cell begins cytokinesis. In animal cells, the cell
pinches inward where the imaginary line used to be (the area of the cell membrane
that pinches to form the two daughter cells is called the cleavage furrow),
separating the two developing nuclei. In plant cells, the daughter cells will
construct a new dividing cell wall between each other. Eventually, the parent cell
will be split in half, giving rise to two daughter cells, each with a replica of the
original genome.
Prokaryotic cells undergo a process similar to mitosis called binary fission.
However, the process of binary fission is very much different from the process of
mitosis, because of the non-involvement of nuclear dynamics and lack of linear
chromosomes.
2.2 Phases of cell cycle and mitosis Interphase
Fig 4 The cell cycle
The mitotic phase is a relatively short period of the cell cycle. It alternates
with the much longer interphase, where the cell prepares itself for cell division.
Interphase is divided into three phases: G1 (first gap), S (synthesis), and G2 (second
gap). During all three phases, the cell grows by producing proteins and
cytoplasmic organelles. However, chromosomes are replicated only during the S
phase. Thus, a cell grows (G1), continues to grow as it duplicates its chromosomes
(S), grows more and prepares for mitosis (G2), and finally it divides (M) before
restarting the cycle. All these phases in the interphase are highly regulated, mainly
via proteins. The phases follow one another in strict order and there are
"checkpoints" that give the cell the cues to proceed from one phase to another.
2.2.1 Preprophase
In plant cells only, prophase is preceded by a pre-prophase stage. In highly
vacuolated plant cells, the nucleus has to migrate into the center of the cell before
mitosis can begin. This is achieved through the formation of a phragmosome, a
transverse sheet of cytoplasm that bisects the cell along the future plane of cell
division. In addition to phragmosome formation, preprophase is characterized by
the formation of a ring of microtubules and actin filaments (called preprophase
band) underneath the plasma membrane around the equatorial plane of the future
mitotic spindle. This band marks the position where the cell will eventually divide.
The cells of higher plants (such as the flowering plants) lack centrioles; instead,
microtubules form a spindle on the surface of the nucleus and are then organized
into a spindle by the chromosomes themselves, after the nuclear membrane breaks
down. The preprophase band disappears during nuclear envelope disassembly and
spindle formation in prometaphase.
Metaphase: The chromosomes have aligned at the metaphase plate.
Prophase: The two round objects above the nucleus are the centrosomes. The chromatin has condensed.
Prometaphase: The nuclear membrane has degraded, and microtubules have invaded the nuclear space. These microtubules can attach to kinetochores or they can interact with opposing microtubules.
Early anaphase: The kinetochore microtubules shorten.
Telophase: The decondensing chromosomes are surrounded by nuclear membranes. Cytokinesis has already begun; the pinched area is known as the cleavage furrow.
Prophase
Fig 5 Micrograph showing condensed chromosomes in blue and the mitotic
spindle in green during prometaphase of mitosis
Normally, the genetic material in the nucleus is in a loosely bundled coil
called chromatin. At the onset of prophase, chromatin condenses together into a
highly ordered structure called a chromosome. Since the genetic material has
already been duplicated earlier in S phase, the replicated chromosomes have two
sister chromatids, bound together at the centromere by the cohesin protein
complex. Chromosomes are typically visible at high magnification through a light
microscope.
Close to the nucleus are structures called centrosomes, which are made of a
pair of centrioles found in most eukaryotic animal cells. The centrosome is the
coordinating center for the cell's microtubules. A cell inherits a single centrosome
at cell division, which is replicated by the cell with the help of the nucleus before a
new mitosis begins, giving a pair of centrosomes. The two centrosomes nucleate
microtubules (which may be thought of as cellular ropes or poles) to form the
spindle by polymerizing soluble tubulin. Molecular motor proteins then push the
centrosomes along these microtubules to opposite sides of the cell. Although
centrioles help organize microtubule assembly, they are not essential for the
formation of the spindle, since they are absent from plants, and centrosomes are
not always used in mitosis.
2.2.2 Prometaphase
The nuclear envelope disassembles and microtubules invade the nuclear
space. This is called open mitosis, and it occurs in most multicellular organisms.
Fungi and some protists, such as algae or trichomonads, undergo a variation called
closed mitosis where the spindle forms inside the nucleus, or its microtubules are
able to penetrate an intact nuclear envelope.
Each chromosome forms two kinetochores at the centromere, one attached at
each chromatid. A kinetochore is a complex protein structure that is analogous to a
ring for the microtubule hook; it is the point where microtubules attach themselves
to the chromosome ( about 1-40 in number, on an average 20 ). Although the
kinetochore structure and function are not fully understood, it is known that it
contains some form of molecular motor. When a microtubule connects with the
kinetochore, the motor activates, using energy from ATP to "crawl" up the tube
toward the originating centrosome. This motor activity, coupled with
polymerisation and depolymerisation of microtubules, provides the pulling force
necessary to later separate the chromosome's two chromatids.
When the spindle grows to sufficient length, kinetochore microtubules begin
searching for kinetochores to attach to. A number of nonkinetochore microtubules
find and interact with corresponding nonkinetochore microtubules from the
opposite centrosome to form the mitotic spindle. Prometaphase is sometimes
considered part of prophase.
In the fishing pole analogy, the kinetochore would be the "hook" that catches
a sister chromatid or "fish". The centrosome acts as the "reel" that draws in the
spindle fibers or "fishing line". It is also one of the main phases of mitosis because
without it cytokinesis would not be able to occur.
2.3 Metaphase
A cell in late metaphase. All chromosomes (blue) but one have arrived at the metaphase plate.
Metaphase comes from the Greek meaning "after." Microtubules find and
attach to kinetochores in prometaphase. Then the two centrosomes start pulling the
chromosomes through their attached centromeres towards the two ends of the cell.
As a result, the chromosomes come under longitudinal tension from the two ends
of the cell. The centromeres of the chromosomes, in some sense, convene along the
metaphase plate or equatorial plane, an imaginary line that is equidistant from the
two centrosome poles. This even alignment is due to the counterbalance of the
pulling powers generated by the opposing kinetochores, analogous to a tug-of-war
between people of equal strength. In certain types of cells, chromosomes do not
line up at the metaphase plate and instead move back and forth between the poles
randomly, only roughly lining up along the midline.
Because proper chromosome separation requires that every kinetochore be
attached to a bundle of microtubules (spindle fibres), it is thought that unattached
kinetochores generate a signal to prevent premature progression to anaphase
without all chromosomes being aligned. The signal creates the mitotic spindle
checkpoint.
2.4 Anaphase
When every kinetochore is attached to a cluster of microtubules and the
chromosomes have lined up along the metaphase plate, the cell proceeds to
anaphase (from the Greek meaning “up,” “against,” “back,” or “re-”).
Two events then occur: first, the proteins that bind sister chromatids together
are cleaved, allowing them to separate. These sister chromatids, which have now
become distinct sister chromosomes, are pulled apart by shortening kinetochore
microtubules and move toward the respective centrosomes to which they are
attached. Next, the nonkinetochore microtubules elongate, pulling the centrosomes
(and the set of chromosomes to which they are attached) apart to opposite ends of
the cell. The force that causes the centrosomes to move towards the ends of the cell
is still unknown, although there is a theory that suggests that the rapid assembly
and breakdown of microtubules may cause this movement.
These two stages are sometimes called early and late anaphase. Early
anaphase is usually defined as the separation of the sister chromatids, while late
anaphase is the elongation of the microtubules and the chromosomes being pulled
farther apart. At the end of anaphase, the cell has succeeded in separating identical
copies of the genetic material into two distinct populations.
2.5 Telophase
Telophase (from the Greek meaning "end") is a reversal of prophase and
prometaphase events. It "cleans up" the after effects of mitosis. At telophase, the
nonkinetochore microtubules continue to lengthen, elongating the cell even more.
Corresponding sister chromosomes attach at opposite ends of the cell. A new
nuclear envelope, using fragments of the parent cell's nuclear membrane, forms
around each set of separated sister chromosomes. Both sets of chromosomes, now
surrounded by new nuclei, unfold back into chromatin. Mitosis is complete, but
cell division is not yet complete.
2.5 Cytokinesis
Cilliate undergoing cytokinesis, with the cleavage furrow being clearly visible
Cytokinesis is often mistakenly thought to be the final part of telophase;
however, cytokinesis is a separate process that begins at the same time as
telophase. Cytokinesis is technically not even a phase of mitosis, but rather a
separate process, necessary for completing cell division. In animal cells, a cleavage
furrow (pinch) containing a contractile ring develops where the metaphase plate
used to be, pinching off the separated nuclei. In both animal and plant cells, cell
division is also driven by vesicles derived from the Golgi apparatus, which move
along microtubules to the middle of the cell. In plants this structure coalesces into a
cell plate at the center of the phragmoplast and develops into a cell wall, separating
the two nuclei. The phragmoplast is a microtubule structure typical for higher
plants, whereas some green algae use a phycoplast microtubule array during
cytokinesis. Each daughter cell has a complete copy of the genome of its parent
cell. The end of cytokinesis marks the end of the M-phase.
2.5.1Significance
Mitosis is important for the maintenance of the chromosomal set; each cell
formed receives chromosomes that are alike in composition and equal in number to
the chromosomes of the parent cell.
Following are the occasions in the lives of organism where mitosis happens:
2.5.2 Development and growth
The number of cells within an organism increases by mitosis. This is the
basis of the development of a multicellular body from a single cell i.e.,
zygote and also the basis of the growth of a multicellular body.
2.5.3 Cell replacement
In some parts of body, e.g. skin and digestive tract, cells are constantly
sloughed off and replaced by new ones. New cells are formed by mitosis and
so are exact copies of the cells being replaced. Similarly, RBCs have short
life span (only about 4 months) and new RBCs are formed by mitosis.
2.5.4 Regeneration
Some organisms can regenerate their parts of bodies. The production of new
cells is achieved by mitosis. For example; sea star regenerates its lost arm
through mitosis.
2.5.6 Asexual reproduction
Some organisms produce genetically similar offspring through asexual
reproduction. For example, the hydra reproduces asexually by budding. The
cells at the surface of hydra undergo mitosis and form a mass called bud.
Mitosis continues in the cells of bud and it grows into a new individual. The
same division happens during asexual reproduction or vegetative
propagation in plants.
2.5.7 Consequences of errors
Although errors in mitosis are rare, the process may go wrong, especially
during early cellular divisions in the zygote. Mitotic errors can be especially
dangerous to the organism because future offspring from this parent cell will carry
the same disorder.
In non-disjunction, a chromosome may fail to separate during anaphase. One
daughter cell will receive both sister chromosomes and the other will receive none.
This results in the former cell having three chromosomes containing the same
genes (two sisters and a homologue), a condition known as trisomy, and the latter
cell having only one chromosome (the homologous chromosome), a condition
known as monosomy. These cells are considered aneuploid, a condition often
associated with cancer. Occasionally when cells experience nondisjunction, they
fail to complete cell division and retain both nuclei in one cell, resulting in
binucleated cells.
Mitosis is a demanding process for the cell, which goes through dramatic
changes in ultrastructure, its organelles disintegrate and reform in a matter of
hours, and chromosomes are jostled constantly by probing microtubules.
Occasionally, chromosomes may become damaged. An arm of the chromosome
may be broken and the fragment lost, causing deletion. The fragment may
incorrectly reattach to another, non-homologous chromosome, causing
translocation. It may reattach to the original chromosome, but in reverse
orientation, causing inversion. Or, it may be treated erroneously as a separate
chromosome, causing chromosomal duplication. The effect of these genetic
abnormalities depends on the specific nature of the error. Errors in the control of
mitosis may cause cancer. All cells have genes that control the timing and number
of mitosis. sometimes mutuations occur in such genes and cells continue to divide.
It results in abnormal cell growth.
Now what happens is that cell abnormally continue to divide at a single
place. It results in the synthesis of execessive tissue growths. When tissues more
than the requirement are synthesized in a single organ, it results in the formation of
Tumors. As long as these tumours remain in their original location they are called
benign tumours. Benign tumours are not harmful as soon as they are not moving.
As soon as they start to move and invade other cells there are said to be malignant
tumours. Malignant tumors are also known as cancerous tumours and their cells are
called cancerous tumours. Such tumours can send cancer cells to other parts in
body where new tumours may form. This phenomenon is called metastasis or
spreading of disease.
2.6 Endomitosis
Endomitosis is a variant of mitosis without nuclear or cellular division,
resulting in cells with many copies of the same chromosome occupying a single
nucleus. This process may also be referred to as endoreduplication and the cells as
endoploid. An example of a cell that goes through endomitosis is the
megakaryocyte.
2.7 Metaphase
Metaphase, from the ancient Greek(between) and (stage), is a stage of
mitosis in the eukaryotic cell cycle in which condensed & highly coiled
chromosomes, carrying genetic information, align in the middle of the cell before
being separated into each of the two daughter cells. Metaphase accounts for
approximately 4% of the cell cycle's duration. Preceded by events in prometaphase
and followed by anaphase, microtubules formed in prophase have already found
and attached themselves to kinetochores in metaphase. The centromeres of the
chromosomes convene themselves on the metaphase plate (or equatorial plate), an
imaginary line that is equidistant from the two centrosome poles.
This even alignment is due to the counterbalance of the pulling powers
generated by the opposing kinetochores, analogous to a tug of war between equally
strong people. In certain types of cells, chromosomes do not line up at the
metaphase plate and instead move back and forth between the poles randomly,
only roughly lining up along the middleline. Early events of metaphase can
coincide with the later events of prometaphase, as chromosomes with connected
kinetochores will start the events of metaphase individually before other
chromosomes with unconnected kinetochores that are still lingering in the events
of prometaphase.
One of the cell cycle checkpoints occurs during prometaphase and
metaphase. Only after all chromosomes have become aligned at the metaphase
plate, when every kinetochore is properly attached to a bundle of microtubules,
does the cell enter anaphase. It is thought that unattached or improperly attached
kinetochores generate a signal to prevent premature progression to anaphase, even
if most of the kinetochores have been attached and most of the chromosomes have
been aligned. Such a signal creates the mitotic spindle checkpoint. This would be
accomplished by regulation of the anaphase-promoting complex, securin, and
separase.
2.8 Metaphase in cytogenetics and cancer studies
The analysis of metaphase chromosomes is one of the main tools of classical
cytogenetics and cancer studies. Chromosomes are condensed(Thickened) and
highly coiled in metaphase, which makes them most suitable for visual analysis.
Metaphase chromosomes make the classical picture of chromosomes (karyotype).
For classical cytogenetic analyses, cells are grown in short term culture and
arrested in metaphase using mitotic inhibitor. Further they are used for slide
preparation and banding (staining) of chromosomes to be visualised under
microscope to study structure and number of chromosomes (karyotype). Staining
of the slides, often with Giemsa (G banding) or Quinacrine, produces a pattern of
in total up to several hundred bands. Normal metaphase spreads are used in
methods like FISH and as a hybridization matrix for comparative genomic
hybridization (CGH) experiments.
Malignant cells from solid tumors or leukemia samples can also be used for
cytogenetic analysis to generate metaphase preparations. Inspection of the stained
metaphase chromosomes allows the determination of numerical and structural
changes in the tumor cell genome, for example, losses of chromosomal segments
or translocations, which may lead to chimeric oncogenes, such as bcr-abl in
chronic myelogenous leukemia.
CHAPTER 3
KARYOTYPING
A karyotype is the number and appearance of chromosomes in the nucleus
of an eukaryotic cell. The term is also used for the complete set of chromosomes in
a species, or an individual organism. Karyotypes describe the number of
chromosomes, and what they look like under a light microscope. Attention is paid
to their length, the position of the centromeres, banding pattern, any differences
between the sex chromosomes, and any other physical characteristics.[4] The
preparation and study of karyotypes is part of cytogenetics. Karyogram of human
male using Giemsa staining. The study of whole sets of chromosomes is
sometimes known as karyology.
The chromosomes are depicted (by rearranging a microphotograph) in a
standard format known as a karyogram or idiogram: in pairs, ordered by size and
position of centromere for chromosomes of the same size. The basic number of
chromosomes in the somatic cells of an individual or a species is called the somatic
number and is designated 2n. Thus, in humans 2n = 46. In the germ-line (the sex
cells) the chromosome number is n (humans: n = 23). So, in normal diploid
organisms, autosomal chromosomes are present in two copies. There may, or may
not, be sex chromosomes. Polyploid cells have multiple copies of chromosomes
and haploid cells have single copies. The study of karyotypes is important for cell
biology and genetics, and the results may be used in evolutionary biology and
medicine. Karyotypes can be used for many purposes; such as, to study
chromosomal aberrations, cellular function, taxonomic relationships, and to gather
information about past evolutionary events.
3.1 History of karyotype studies
Chromosomes were first observed in plant cells by Karl Wilhelm von Nägeli
in 1842. Their behavior in animal (salamander) cells was described by Walther
Flemming, the discoverer of mitosis, in 1882. The name was coined by another
German anatomist, von Waldeyer in 1888. The next stage took place after the
development of genetics in the early 20th century, when it was appreciated that the
set of chromosomes (the karyotype) was the carrier of the genes. Levitsky seems to
have been the first to define the karyotype as the phenotypic appearance of the
somatic chromosomes, in contrast to their genic contents.
The subsequent history of the concept can be followed in the works of
Darlington and White. Investigation into the human karyotype took many years to
settle the most basic question: how many chromosomes does a normal diploid
human cell contain? In 1912, Hans von Winiwarter reported 47 chromosomes in
spermatogonia and 48 in oogonia, concluding an XX/XO sex determination
mechanism. Painter in 1922 was not certain whether the diploid number of humans
was 46 or 48, at first favoring 46. He revised his opinion later from 46 to 48, and
he correctly insisted on humans having an XX/XY system. Considering their
techniques, these results were quite remarkable.
New techniques were needed to definitively solve the problem:
1. Using cells in culture
2. Pretreating cells in a hypotonic solution, which swells them and spreads the
chromosomes
3. Arresting mitosis in metaphase by a solution of colchicines
4. Squashing the preparation on the slide forcing the chromosomes into a
single plane
5. Cutting up a photomicrograph and arranging the result into an indisputable
karyogram.
It took until the mid 1950s until it became generally accepted that the karyotype
of humans included only 46 chromosomes. Rather interestingly, the great apes
have 48 chromosomes. Human chromosome 2 was formed by a merger of ancestral
chromosomes, reducing the number.
3.2 Observations on karyotypes
3.2.1 Staining
The study of karyotypes is made possible by staining. Usually, a
suitable dye, such as Giemsa, is applied after cells have been arrested during cell
division by a solution of colchicine. For humans, white blood cells are used most
frequently because they are easily induced to divide and grow in tissue culture.
[16] Sometimes observations may be made on non-dividing (interphase) cells. The
sex of an unborn fetus can be determined by observation of interphase cells.
3.2.2 Observations
Six different characteristics of karyotypes are usually observed and
compared:
1. Differences in absolute sizes of chromosomes. Chromosomes can vary in
absolute size by as much as twenty-fold between genera of the same
family: Lotus tenuis and Vicia faba (legumes), both have six pairs of
chromosomes (n=6) yet V. faba chromosomes are many times larger. This
feature probably reflects different amounts of DNA duplication.
2. Differences in the position of centromeres. This is brought about
by translocations.
3. Differences in relative size of chromosomes can only be caused by
segmental interchange of unequal lengths.
4. Differences in basic number of chromosomes may occur due to successive
unequal translocations which finally remove all the essential genetic
material from a chromosome, permitting its loss without penalty to the
organism (the dislocation hypothesis). Humans have one pair fewer
chromosomes than the great apes, but the genes have been mostly
translocated (added) to other chromosomes.
5. Differences in number and position of satellites, which (when they occur)
are small bodies attached to a chromosome by a thin thread.
6. Differences in degree and distribution of heterochromatic regions.
Heterochromatin stains darker than euchromatin, indicating tighter packing,
and mainly consists of genetically inactive repetitive DNA sequences.
A full account of a karyotype may therefore include the number, type,
shape and banding of the chromosomes, as well as other cytogenetic
information.
Variation is often found:
1. Between the sexes
2. Between the germ-line and soma (between gametes and the rest of the body)
3. Between members of a population (chromosome polymorphism)
4. Geographical variation between races
5. Mosaics or otherwise abnormal individuals.
3.3 The human karyotype
Most (but not all) species have a standard karyotype. The normal human
karyotypes contain 22 pairs of autosomal chromosomes and one pair of sex
chromosomes. Normal karyotypes for females contain two X chromosomes and are
denoted 46, XX; males have both an X and a Y chromosome denoted 46, XY. Any
variation from the standard karyotype may lead to developmental abnormalities.
Fig Diversity and evolution of karyotype
Although the replication and transcription of DNA is highly standardized
in eukaryotes, the same cannot be said for their karyotypes, which are highly
variable. There is variation between species in chromosome number, and in
detailed organization, despite their construction from the same macromolecules.
This variation provides the basis for a range of studies in evolutionary cytology.
In some cases there is even significant variation within species. In a review,
Godfrey and Masters conclude: "In our view, it is unlikely that one process or the
other can independently account for the wide range of karyotype structures that are
observed... But, used in conjunction with other phylogenetic data, karyotypic
fissioning may help to explain dramatic differences in diploid numbers between
closely related species, which were previously inexplicable.
Although much is known about karyotypes at the descriptive level, and it is
clear that changes in karyotype organization have had effects on the evolutionary
course of many species, it is quite unclear what the general significance might be.
"We have a very poor understanding of the causes of karyotype evolution; despite
many careful investigations... the general significance of karyotype evolution is
obscure.
3.3.1 Changes during development
Instead of the usual gene repression, some organisms go in for large-scale
elimination of heterochromatin, or other kinds of visible adjustment to the
karyotype. Chromosome elimination. In some species, as in many sciarid flies,
entire chromosomes are eliminated during development. Chromatin diminution
(founding father: Theodor Boveri). In this process, found in some copepods and
roundworms such as Ascaris suum, portions of the chromosomes are cast away in
particular cells. This process is a carefully organised genome rearrangement where
new telomeres are constructed and certain heterochromatin regions are lost. In A.
suum, all the somatic cell precursors undergo chromatin diminution. X-
inactivation.
The inactivation of one X chromosome takes place during the early
development of mammals (see Barr body and dosage compensation). In placental
mammals, the inactivation is random as between the two Xs; thus the mammalian
female is a mosaic in respect of her X chromosomes. In marsupials it is always the
paternal X which is inactivated. In human females some 15% of somatic cells
escape inactivation.
3.3.2 Number of chromosomes in a set
A spectacular example of variability between closely related species is the
muntjac, which was investigated by Kurt Benirschke and his colleague Doris
Wurster. The diploid number of the Chinese muntjac, Muntiacus reevesi, was
found to be 46, all telocentric. When they looked at the karyotype of the closely
related Indian muntjac, Muntiacus muntjak, they were astonished to find it had
female = 6, male = 7 chromosomes. "They simply could not believe what they
saw... They kept quiet for two or three years because they thought something was
wrong with their tissue culture... But when they obtained a couple more specimens
they confirmed [their findings]" Hsu p73-4 The number of chromosomes in the
karyotype between (relatively) unrelated species is hugely variable.
The low record is held by the nematode Parascaris univalens, where the
haploid n = 1; the high record would be somewhere amongst the ferns, with the
Adder's Tongue Fern Ophioglossum ahead with an average of 1262 chromosomes.
Top score for animals might be the shortnose sturgeon Acipenser brevirostrum at a
mere 372 chromosomes. The existence of supernumerary or B chromosomes
means that chromosome number can vary even within one interbreeding
population; and aneuploids are another example, though in this case they would not
be regarded as normal members of the population.
3.3.3 Fundamental number
The fundamental number, FN, of a karyotype is the number of visible major
chromosomal arms per set of chromosomes. Thus, FN ≤ 2n, the difference
depending on the number of chromosomes considered single-armed (acrocentric or
telocentric) present. Humans have FN = 82, due to the presence of five acrocentric
chromosome pairs (13, 14, 15, 21 and 22).
3.4 Ploidy
Ploidy is the number of complete sets of chromosomes in a cell. Polyploidy, where
there are more than two sets of homologous chromosomes in the cells, occurs
mainly in plants. It has been of major significance in plant evolution according to
Stebbins. The proportion of flowering plants which are polyploid was estimated by
Stebbins to be 30-35%, but in grasses the average is much higher, about 70%.
Polyploidy in lower plants (ferns, horsetails and psilotales) is also common, and
some species of ferns have reached levels of polyploidy far in excess of the highest
levels known in flowering plants.
Polyploidy in animals is much less common, but it has been significant in some
groups. Polyploid series in related species which consist entirely of multiples of a
single basic number are known as euploid. Haplo-diploidy, where one sex is
diploid, and the other haploid. It is a common arrangement in the Hymenoptera,
and in some other groups.Endopolyploidy occurs when in adult differentiated
tissues the cells have ceased to divide by mitosis, but the nuclei contain more than
the original somatic number of chromosomes. In the endocycle (endomitosis or
endoreduplication) chromosomes in a 'resting' nucleus undergo reduplication, the
daughter chromosomes separating from each other inside an intact nuclear
membrane.
In many instances, endopolyploid nuclei contain tens of thousands of
chromosomes (which cannot be exactly counted). The cells do not always contain
exact multiples (powers of two), which is why the simple definition 'an increase in
the number of chromosome sets caused by replication without cell division' is not
quite accurate.
This process (especially studied in insects and some higher plants such as maize)
may be a developmental strategy for increasing the productivity of tissues which
are highly active in biosynthesis.
The phenomenon occurs sporadically throughout the eukaryote kingdom from
protozoa to man; it is diverse and complex, and serves differentiation and
morphogenesis in many ways. See palaeopolyploidy for the investigation of
ancient karyotype duplications.
3.5 Aneuploidy
Aneuploidy is the condition in which the chromosome number in the cells is
not the typical number for the species. This would give rise to a chromosome
abnormality such as an extra chromosome or one or more chromosomes lost.
Abnormalities in chromosome number usually cause a defect in development.
Down syndrome and Turner syndrome are examples of this.
Aneuploidy may also occur within a group of closely related species. Classic
examples in plants are the genus Crepis, where the gametic (= haploid) numbers
form the series x = 3, 4, 5, 6, and 7; and Crocus, where every number from x = 3 to
x = 15 is represented by at least one species. Evidence of various kinds shows that
that trends of evolution have gone in different directions in different groups.[41]
Closer to home, the great apes have 24x2 chromosomes whereas humans have
23x2. Human chromosome 2 was formed by a merger of ancestral chromosomes,
reducing the number.
3.5 Chromosomal polymorphism
Some animal species are polymorphic for chromosome fusions or
dissociations. When this happens, the chromosome number is variable from one
individual to another. Well-researched examples are the ladybird beetle Chilocorus
stigma, some mantids of the genus Ameles, the European shrew Sorex araneus.
There is some evidence from the case of the mollusc Thais lapillus (the dog whelk)
on the Brittany coast, that the two chromosome morphs are adapted to different
habitats.
3.6 Species trees
The detailed study of chromosome banding in insects with polytene
chromosomes can reveal relationships between closely related species: the classic
example is the study of chromosome banding in Hawaiian drosophilids by
Hampton Carson.
In about 6,500 sq mi (17,000 km2), the Hawaiian Islands have the most
diverse collection of drosophilid flies in the world, living from rainforests to
subalpine meadows. These roughly 800 Hawaiian drosophilid species are usually
assigned to two genera, Drosophila and Scaptomyza, in the family Drosophilidae.
The polytene banding of the 'picture wing' group, the best-studied group of
Hawaiian drosophilids, enabled Carson to work out the evolutionary tree long
before genome analysis was practicable. In a sense, gene arrangements are visible
in the banding patterns of each chromosome. Chromosome rearrangements,
especially inversions, make it possible to see which species are closely related.
The results are clear. The inversions, when plotted in tree form (and
independent of all other information), show a clear "flow" of species from older to
newer islands. There are also cases of colonization back to older islands, and
skipping of islands, but these are much less frequent. Using K-Ar dating, the
present islands date from 0.4 million years ago (mya) (Mauna Kea) to 10mya
(Necker). The oldest member of the Hawaiian archipelago still above the sea is
Kure Atoll, which can be dated to 30 mya. The archipelago itself (produced by the
Pacific plate moving over a hot spot) has existed for far longer, at least into the
Cretaceous. Previous islands now beneath the sea (guyots) form the Emperor
Seamount Chain.
All of the native Drosophila and Scaptomyza species in Hawaii have
apparently descended from a single ancestral species that colonized the islands,
probably 20 million years ago. The subsequent adaptive radiation was spurred by a
lack of competition and a wide variety of niches. Although it would be possible for
a single gravid female to colonise an island, it is more likely to have been a group
from the same species.
There are other animals and plants on the Hawaiian archipelago which
have undergone similar, if less spectacular, adaptive radiations.
3.7 Depiction of karyotypes
3.7.1 Types of banding
Cytogenetics employs several techniques to visualize different aspects of
chromosomes:
G-banding is obtained with Giemsa stain following digestion of
chromosomes with trypsin. It yields a series of lightly and darkly stained
bands - the dark regions tend to be heterochromatic, late-replicating and AT
rich. The light regions tend to be euchromatic, early-replicating and GC rich.
This method will normally produce 300-400 bands in a normal, human
genome.
R-banding is the reverse of G-banding (the R stands for "reverse"). The dark
regions are euchromatic (guanine-cytosine rich regions) and the bright
regions are heterochromatic (thymine-adenine rich regions).
C-banding: Giemsa binds to constitutive heterochromatin, so it stains
centromeres.
Q-banding is a fluorescent pattern obtained using quinacrine for staining.
The pattern of bands is very similar to that seen in G-banding.
T-banding: visualize telomeres.
Silver staining: Silver nitrate stains the nucleolar organization region-
associated protein. This yields a dark region where the silver is deposited,
denoting the activity of rRNA genes within the NOR.
3.7.2 Classic karyotype cytogenetics
Karyogram from a human female lymphocyte probed for the Alu sequence using
FISH. In the "classic" (depicted) karyotype, a dye, often Giemsa (G-banding), less
frequently Quinacrine, is used to stain bands on the chromosomes. Giemsa is
specific for the phosphate groups of DNA. Quinacrine binds to the adenine-
thymine-rich regions. Each chromosome has a characteristic banding pattern that
helps to identify them; both chromosomes in a pair will have the same banding
pattern.
Karyotypes are arranged with the short arm of the chromosome on top, and
the long arm on the bottom. Some karyotypes call the short and long arms p and q,
respectively. In addition, the differently stained regions and sub-regions are given
numerical designations from proximal to distal on the chromosome arms. For
example, Cri du chat syndrome involves a deletion on the short arm of
chromosome 5. It is written as 46,XX,5p-. The critical region for this syndrome is
deletion of 15.2, which is written as 46,XX,del(5)(p15.2)
3.7.3 Spectral karyotype (SKY technique)
Spectral karyotyping is a molecular cytogenetic technique used to
simultaneously visualize all the pairs of chromosomes in an organism in different
colors. Fluorescently labeled probes for each chromosome are made by labeling
chromosome-specific DNA with different fluorophores. Because there are a
limited number of spectrally-distinct fluorophores, a combinatorial labeling
method is used to generate many different colors. Spectral differences generated by
combinatorial labeling are captured and analyzed by using an interferometer
attached to a fluorescence microscope. Image processing software then assigns a
pseudo color to each spectrally different combination, allowing the visualization of
the individually colored chromosomes.
This technique is used to identify structural chromosome aberrations in cancer cells
and other disease conditions when Giemsa banding or other techniques are not
accurate enough.
3.8 Digital karyotyping
Digital karyotyping is a technique used to quantify the DNA copy number on
a genomic scale. Short sequences of DNA from specific loci all over the genome
are isolated and enumerated. This method is also known as virtual karyotyping.
CHAPTER 4
4.1 CHROMOSOMAL ABNORMALITIES
Chromosome abnormalities can be numerical, as in the presence of extra or
missing chromosomes, or structural, as in derivative chromosome, translocations,
inversions, large-scale deletions or duplications. Numerical abnormalities, also
known as aneuploidy, often occur as a result of nondisjunction during meiosis in
the formation of a gamete; trisomies, in which three copies of a chromosome are
present instead of the usual two, are common numerical abnormalities. Structural
abnormalities often arise from errors in homologous recombination. Both types of
abnormalities can occur in gametes and therefore will be present in all cells of an
affected person's body, or they can occur during mitosis and give rise to a genetic
mosaic individual who has some normal and some abnormal cells.
Chromosomal abnormalities that lead to disease in humans include
Turner syndrome results from a single X chromosome (45, X or 45, X0).
Klinefelter syndrome , the most common male chromosomal disease,
otherwise known as 47, XXY is caused by an extra X chromosome.
Edwards syndrome is caused by trisomy (three copies) of chromosome 18.
Down syndrome , a common chromosomal disease, is caused by trisomy of
chromosome 21.
Patau syndrome is caused by trisomy of chromosome 13.
Also documented are trisomy 8, trisomy 9 and trisomy 16, although they
generally do not survive to birth.
Some disorders arise from loss of just a piece of one chromosome, including
Cri du chat (cry of the cat), from a truncated short arm on chromosome 5.
The name comes from the babies' distinctive cry, caused by abnormal
formation of the larynx.
1p36 Deletion syndrome , from the loss of part of the short arm of
chromosome 1.
Angelman syndrome – 50% of cases have a segment of the long arm of
chromosome 15 missing; a deletion of the maternal genes, example of
imprinting disorder.
Prader-Willi syndrome – 50% of cases have a segment of the long arm of
chromosome 15 missing; a deletion of the paternal genes, example of
imprinting disorder.
Chromosomal abnormalities can also occur in cancerous cells of an otherwise
genetically normal individual; one well-documented example is the Philadelphia
chromosome, a translocation mutation commonly associated with chronic
myelogenous leukemia and less often with acute lymphoblastic leukemia.
A chromosome anomaly, abnormality or aberration reflects an atypical
number of chromosomes or a structural abnormality in one or more chromosomes.
A Karyotype refers to a full set of chromosomes from an individual which can be
compared to a "normal" Karyotype for the species via genetic testing.
A chromosome anomaly may be detected or confirmed in this manner.
Chromosome anomalies usually occur when there is an error in cell division
following meiosis or mitosis. There are many types of chromosome anomalies.
They can be organized into two basic groups, numerical and structural anomalies.
4.2 Numerical Disorders
This is called Aneuploidy (an abnormal number of chromosomes), and
occurs when an individual is missing either a chromosome from a pair
(monosomy) or has more than two chromosomes of a pair (Trisomy, Tetrasomy,
etc.). In humans an example of a condition caused by a numerical anomaly is
Down Syndrome, also known as Trisomy 21 (an individual with Down Syndrome
has three copies of chromosome 21, rather than two). Turner Syndrome is an
example of a monosomy where the individual is born with only one sex
chromosome, an X.
4.3 Structural abnormalities
When the chromosome's structure is altered. This can take several forms:
Deletions : A portion of the chromosome is missing or deleted. Known
disorders in humans include Wolf-Hirschhorn syndrome, which is caused by
partial deletion of the short arm of chromosome 4; and Jacobsen syndrome,
also called the terminal 11q deletion disorder.
Duplications : A portion of the chromosome is duplicated, resulting in extra
genetic material. Known human disorders include Charcot-Marie-Tooth
disease type 1A which may be caused by duplication of the gene encoding
peripheral myelin protein 22 (PMP22) on chromosome 17.
Translocations : When a portion of one chromosome is transferred to
another chromosome. There are two main types of translocations. In a
reciprocal translocation, segments from two different chromosomes have
been exchanged. In a Robertsonian translocation, an entire chromosome has
attached to another at the Centromere - in humans these only occur with
chromosomes 13, 14, 15, 21 and 22.
Inversions : A portion of the chromosome has broken off, turned upside
down and reattached, therefore the genetic material is inverted.
Rings : A portion of a chromosome has broken off and formed a circle or
ring. This can happen with or without loss of genetic material.
Isochromosome : Formed by the mirror image copy of a chromosome
segment including the centromere.
Chromosome instability syndromes are a group of disorders characterized by
chromosomal instability and breakage. They often lead to an increased tendency to
develop certain types of malignancies.
4.3 Inheritance
Most chromosome abnormalities occur as an accident in the egg or sperm,
and are therefore initially not inherited. Therefore, the anomaly is present in every
cell of the body. Some anomalies, however, can happen after conception, resulting
in Mosaicism (where some cells have the anomaly and some do not). Chromosome
anomalies can be inherited from a parent or be "de novo". This is why
chromosome studies are often performed on parents when a child is found to have
an anomaly.
4.4 Cytogenetics
Cytogenetics is a branch of genetics that is concerned with the study of the
structure and function of the cell, especially the chromosomes. It includes routine
analysis of G-Banded chromosomes, other cytogenetic banding techniques, as well
as molecular cytogenetics such as fluorescent in situ hybridization (FISH) and
comparative genomic hybridization (CGH).
4.5 Early years
Chromosomes were first observed in plant cells by Karl Wilhelm von Nägeli
in 1842. Their behavior in animal (salamander) cells was described by Walther
Flemming, the discoverer of mitosis, in 1882. The name was coined by another
German anatomist, von Waldeyer in 1888.
The next stage took place after the development of genetics in the early 20th
century, when it was appreciated that the set of chromosomes (the karyotype) was
the carrier of the genes. Levitsky seems to have been the first to define the
karyotype as the phenotypic appearance of the somatic chromosomes, in contrast
to their genic contents. Investigation into the human karyotype took many years to
settle the most basic question: how many chromosomes does a normal diploid
human cell contain? In 1912, Hans von Winiwarter reported 47 chromosomes in
spermatogonia and 48 in oogonia, concluding an XX/XO sex determination
mechanism. Painter in 1922 was not certain whether the diploid number of man
was 46 or 48, at first favoring 46. He revised his opinion later from 46 to 48, and
he correctly insisted on man having an XX/XY system. Considering their
techniques, these results were quite remarkable.
New techniques were needed to definitively solve the problem:
1. Using cells in culture
2. Pre-treating cells in a hypotonic solution, which swells them and
spreads the chromosomes
3. Arresting mitosis in metaphase by a solution of colchicine
4. Squashing the preparation on the slide forcing the chromosomes into a
single plane
5. Cutting up a photomicrograph and arranging the result into an
indisputable karyogram.
It took until 1956 until it became generally accepted that the karyotype of man
included only 46 chromosomes. Rather interestingly, the great apes have 48
chromosomes. Human chromosome 2 was formed by a merger of ancestral
chromosomes, reducing the number.
4.6 Applications in biology
4.6.1 McClintock's work on maize
Barbara McClintock began her career as a maize cytogeneticist. In 1931,
McClintock and Harriet Creighton demonstrated that cytological recombination of
marked chromosomes correlated with recombination of genetic traits (genes).
McClintock continued her career in cytogenetics studying the mechanics and
inheritance of broken and ring (circular) chromosomes of maize. During her
cytogenetic work, McClintock discovered transposons, a find which eventually led
to her Nobel Prize in 1983.
4.6.2 Natural populations of Drosophila
In the 1930s, Dobzhansky and his co-workers collected Drosophila
pseudoobscura and D. persimilis from wild populations in California and
neighboring states. Using Painter's technique they studied the polytene
chromosomes and discovered that the wild populations were polymorphic for
chromosomal inversions. All the flies look alike whatever inversions they carry:
this is an example of a cryptic polymorphism.
Evidence rapidly accumulated to show that natural selection was
responsible. Using a method invented by L'Heretier and Teissier, Dobzhansky bred
populations in population cages, which enabled feeding, breeding and sampling
whilst preventing escape. This had the benefit of eliminating migration as a
possible explanation of the results. Stocks containing inversions at a known initial
frequency can be maintained in controlled conditions. It was found that the various
chromosome types do not fluctuate at random, as they would if selectively neutral,
but adjust to certain frequencies at which they become stabilised. By the time
Dobzhansky published the third edition of his book in 1951 he was persuaded that
the chromosome morphs were being maintained in the population by the selective
advantage of the heterozygotes, as with most polymorphisms.
4.7 Human abnormalities and medical applications
In the event of procedures which allowed easy enumeration of
chromosomes, discoveries were quickly made related to aberrant chromosomes or
chromosome number. In some congenital disorders, such as Down's syndrome,
cytogenetics revealed the nature of the chromosomal defect: a "simple" trisomy.
Abnormalities arising from nondisjunction events can cause cells with aneuploidy
(additions or deletions of entire chromosomes) in one of the parents or in the fetus.
In 1959, Lejeune discovered patients with Down syndrome had an extra copy of
chromosome 21. Down syndrome is also referred to as trisomy 21.
Other numerical abnormalities discovered include sex chromosome
abnormalities. An individual with only one sex chromosome (the X) has Turner
syndrome, an additional X chromosome in a male, resulting in 47 total
chromosomes, has Klinefelter's Syndrome. Many other sex chromosome
combinations are compatible with live birth including XXX, XYY, and XXXX.
The ability for mammals to tolerate aneuploidies in the sex chromosomes arises
from the ability to inactivate them, which is required in normal females to
compensate for having two copies of the chromosome. Not all genes on the X
Chromosome are inactivated, which is why there is a phenotypic effect seen in
individuals with extra X chromosomes.
Trisomy 13 was associated with Patau's Syndrome and trisomy 18 with
Edward's Syndrome.
In 1960, Peter Nowell and David Hungerford[17] discovered a small
chromosome in the white blood cells of patients with Chronic myelogenous
leukemia (CML). This abnormal chromosome was dubbed the Philadelphia
chromosome - as both scientists were doing their research in Philadelphia,
Pennsylvania. Thirteen years later, with the development of more advanced
techniques, the abnormal chromosome was shown by Janet Rowley to be the result
of a translocation of chromosomes 9 and 22. Identification of the Philadelphia
chromosome by cytogenetics, in addition to other tests, is used today as a
diagnostic for CML.
FIG Advent of banding techniques
In the late 1960s, Caspersson developed banding techniques which
differentially stain chromosomes. This allows chromosomes of otherwise equal
size to be differentiated as well as to elucidate the breakpoints and constituent
chromosomes involved in chromosome translocations. Deletions within one
chromosome could also now be more specifically named and understood. Deletion
syndromes such as DiGeorge syndrome, Prader-Willi syndrome and others were
discovered to be caused by deletions in chromosome material.
Diagrams identifying the chromosomes based on the banding patterns are
known as cytogenetic maps. These maps became the basis for both prenatal and
oncological fields to quickly move cytogenetics into the clinical lab where
karyotyping allowed scientists to look for chromosomal alterations. Techniques
were expanded to allow for culture of free amniocytes recovered from amniotic
fluid, and elongation techniques for all culture types that allow for higher
resolution banding.
4.8 Beginnings of molecular cytogenetics
In the 1980s, advances were made in molecular cytogenetics. While
radioisotope-labeled probes had been hybridized with DNA since 1969, movement
was now made in using fluorescent labeled probes. Hybridizing them to
chromosomal preparations using existing techniques came to be known as
fluorescent in situ hybridization (FISH). This change significantly increased the
usage of probing techniques as fluorescent labeled probes are safer and can be used
almost indefinitely. Further advances in micromanipulation and examination of
chromosomes led to the technique of chromosome microdissection whereby
aberrations in chromosomal structure could be isolated, cloned and studied in ever
greater detail.
CHAPTER 5
Techniques
5.1 Karyotyping
Routine chromosome analysis (Karyotyping) refers to analysis of metaphase
chromosomes which have been banded using trypsin followed by Giemsa,
Leishmanns, or a mixture of the two. This creates unique banding patterns on the
chromosomes. The molecular mechanism and reason for these patterns is
unknown, although it likely related to replication timing and chromatin packing.
Several chromosome-banding techniques are used in cytogenetics laboratories.
Quinacrine banding (Q-banding) was the first staining method used to produce
specific banding patterns. This method requires a fluorescence microscope and is
no longer as widely used as Giemsa banding (G-banding). Reverse banding (R-
banding) requires heat treatment and reverses the usual white and black pattern that
is seen in G-bands and Q-bands. This method is particularly helpful for staining the
distal ends of chromosomes. Other staining techniques include C-banding and
nucleolar organizing region stains (NOR stains). These latter methods specifically
stain certain portions of the chromosome.
C-banding stains the constitutive heterochromatin, which usually lies near
the centromere, and NOR staining highlights the satellites and stalks of acrocentric
chromosomes. High-resolution banding involves the staining of chromosomes
during prophase or early metaphase (prometaphase), before they reach maximal
condensation. Because prophase and prometaphase chromosomes are more
extended than metaphase chromosomes, the number of bands observable for all
chromosomes increases from about 300 to 450 to as many as 800. This allows the
detection of less obvious abnormalities usually not seen with conventional
banding.
5.2 Slide preparation
Cells from bone marrow, blood, amniotic fluid, cord blood, tumor, and
tissues (including skin, umbilical cord, chorionic villi, liver, and many other
organs) can be cultured using standard cell culture techniques in order to increase
their number. A mitotic inhibitor (colchicine, colcemid) is then added to the
culture. This stops cell division at mitosis which allows an increased yield of
mitotic cells for analysis. The cells are then centrifuged and media and mitotic
inhibitor are removed, and replaced with a hypotonic solution. This causes the
white blood cells or fibroblasts to swell so that the chromosomes will spread when
added to a slide as well as lyses the red blood cells. After the cells have been
allowed to sit in hypotonic, Carnoy's fixative (3:1 methanol to glacial acetic acid)
is added. This kills the cells and hardens the nuclei of the remaining white blood
cells. The cells are generally fixed repeatedly to remove any debris or remaining
red blood cells. The cell suspension is then dropped onto specimen slides. After
aging the slides in an oven or waiting a few days they are ready for banding and
analysis.
5.3 Analysis
Analysis of banded chromosomes is done at a microscope by a clinical
laboratory specialist in cytogenetics (CLSp(CG)). Generally 20 cells are analyzed
which is enough to rule out mosaicism to an acceptable level. The results are
summarized and given to a board-certified cytogeneticist for review, and to write
an interpretation taking into account the patients previous history and other clinical
findings. The results are then given out reported in an International System for
Human Cytogenetic Nomenclature 2009 (ISCN2009).
5.4 Fluorescent in situ hybridization
Fluorescent in situ hybridization refers to using fluorescently labeled probe to
hybridize to cytogenetic cell preparations.
In addition to standard preparations FISH can also be performed on:
bone marrow smears
blood smears
paraffin embedded tissue preparations
enzymatically dissociated tissue samples
uncultured bone marrow
uncultured amniocytes
cytospin preparations
5.5 Slide preparation
The slide is aged using a salt solution usually consisting of 2X SSC (salt,
sodium citrate). The slides are then dehydrated in ethanol, and the probe mixture is
added. The sample DNA and the probe DNA are then co-denatured using a heated
plate and allowed to re-anneal for at least 4 hours. The slides are then washed to
remove excess unbound probe, and counterstained with 4',6-Diamidino-2-
phenylindole (DAPI) or propidium iodide.
5.7 Analysis
Analysis of FISH specimens is done by fluorescence microscopy by a
clinical laboratory specialist in cytogenetics. For oncology generally a large
number of interphase cells are scored in order to rule out low level residual disease,
generally between 200 and 1000 cells are counted and scored. For congenital
problems usually 20 metaphase cells are scored.
Future of cytogenetics
Advances now focus on molecular cytogenetics including automated
systems for counting the results of standard FISH preparations and techniques for
virtual karyotyping, such as comparative genomic hybridization arrays, CGH and
Single nucleotide polymorphism-arrays.
CHAPTER 6
MATLAB
MATLAB is a high-level technical computing language and interactive
environment for algorithm development, data visualization, data analysis, and
numerical computation. Using MATLAB, you can solve technical computing
problems faster than with traditional programming languages, such as C, C++, and
FORTRAN.
You can use MATLAB in a wide range of applications, including signal and
image processing, communications, control design, test and measurement,
financial modeling and analysis, and computational biology. Add-on toolboxes
(collections of special-purpose MATLAB functions) extend the MATLAB
environment to solve particular classes of problems in these application areas.
MATLAB provides a number of features for documenting and sharing your
work. You can integrate your MATLAB code with other languages and
applications, and distribute your MATLAB algorithms and applications. The
MATLAB language supports the vector and matrix operations that are fundamental
to engineering and scientific problems. It enables fast development and execution.
With the MATLAB language, you can program and develop algorithms
faster than with traditional languages because you do not need to perform low-
level administrative tasks, such as declaring variables, specifying data types, and
allocating memory. In many cases, MATLAB eliminates the need for ‘for’ loops.
As a result, one line of MATLAB code can often replace several lines of C or C++
code.
6.1 Image Processing
The image processing step aims at image contrast enhancement and
compensation of geometric distortions observed in each chromosome not related
with its intrinsic shape or size. The image brightness and contrast depend on the
specific tuning of the microscope and the particular geometric shape of each
chromosome depends on the specific metaphase plaque from which the
chromosomes were extracted. These effects must be compensated to improve the
results of the pairing algorithm.
The image processing step is composed of the following operations.
1) Chromosome extraction—Each chromosome is isolated from the unordered
karyogram.
2) Geometrical compensation—The geometric compensation, performed by
using the algorithm is needed to obtain chromosomes with vertical medial axis,
This compensation algorithm is composed of the following main steps:
a) chromosome and medial axis segmentation
b) axis smoothing
c) interpolationalong orthogonal lines to the smoothed medial axis
d) border regularization
3) Shape normalization—The features used in the comparison of chromosomes
are grouped into two classes:
1) geometric based
2) pattern based (G-banding).
To compare chromosomes from a band pattern point of view, geometrical
and dimensional differences must be removed, or at least attenuated. Therefore, a
dimensional scaling is performed before the pattern features is extracted to make
all the chromosome with the same size and aspect ratio by interpolating the
original images.
4) Intensity compensation—The metaphase plaque from which the chromosomes
are extracted does not present a uniform brightness and contrast. To compensate
for this inhomogeneity, the spatially scaled images are histogram equalized.
6.2 Concepts used in this phase
1) Image conversion
2) Denoising
3) Edge detection
4) Two dimensional convolutions.
6.2.1 Image conversion
The toolbox includes many functions that you can use to convert an image from
one type to another, listed in the following table. For example, if you want to filter
a color image that is stored as an indexed image, you must first convert it to true
color format. When you apply the filter to the true color image, MATLAB filters
the intensity values in the image, as is appropriate. If you attempt to filter the
indexed image, MATLAB simply applies the filter to the indices in the indexed
image matrix, and the results might not be meaningful. You can perform certain
conversions just using MATLAB syntax. For example, you can convert a grayscale
image to true color format by concatenating three copies of the original matrix
along the third dimension.
RGB = cat (3,I,I,I);
The resulting true color image has identical matrices for the red, green, and blue
planes, so the image displays as shades of gray.
In addition to these image type conversion functions, there are other functions
that return a different image type as part of the operation they perform. For
example, the region of interest functions returns a binary image that you can use to
mask an image for filtering or for other operations.
6.2.4 Denoising
We may define noise to be any degradation in the image signal, caused by
external disturbance. If an image is being sent electronically from one place to
another, via satellite or wireless transmission, or through networked cable, we may
expect errors to occur in the image signal. These errors will appear on the image
output in different ways depending on the type of disturbance in the signal.
Usually we know what type of errors to expect, and hence the type of noise on the
image; hence we can choose the most appropriate method for reducing the effects.
Cleaning an image corrupted by noise is thus an important area of image
restoration.
6.2.5 Edge detection
Edges contain some of the most useful information in an image. We may use
edges to measure the size of objects in an image; to isolate particular objects from
their background; to recognize or classify objects. There is a large number of edge
finding algorithms in existence, and we shall look at some of the more
straightforward of them. The general Matlab command for finding edges is
edge(image,'method',parameters. . . ) Where the parameters available depend on
the method used
6.3 Two dimensional convolutions
C = conv2(A,B) computes the two-dimensional convolution of matrices A
and B. If one of these matrices describes a two-dimensional finite impulse response
(FIR) filter, the other matrix is filtered in two dimensions. The size of C in each
dimension is equal to the sum of the corresponding dimensions of the input
matrices, minus one. That is, if the size of A is [ma,na] and the size of B is
[mb,nb], then the size of C is [ma+mb-1,na+nb-1].
The indices of the center element of B are defined as floor(([mb nb]+1)/2).
C = conv2(hcol,hrow,A) convolves A first with the vector hcol along the rows and
then with the vector hrow along the columns. If hcol is a column vector and hrow
is a row vector, this case is the same as C = conv2(hcol*hrow,A). C =
conv2(...,'shape') returns a subsection of the two-dimensional convolution, as
specified by the shape parameter
Algorithms description
1) Read the image and convert into gray
2)Remove noise
3) Background separation
4) Edge detect
5) Separate the pairs
MODULE 1
PSEUDO CODE
iimread('12345.bmp');
rgb2gray
im2bw(im,0.7);
imedfilt2(im1,[3 3]);
edge(im1,'sobel');
[imx,imy]=size(BW);
Msk
conv2(double(BW),double(msk));
bwlabel(B,8);
mx=max(max(L));
[r,c] = find(L==22);
rc = [r c];
[sx sy]=size(rc);
nzeros(imx,imy);
for i=1:sx
x1=rc(i,1);
y1=rc(i,2);
n1(x1,y1)=255;
MODULE 2
clc
[m,n]=size(L);
L_number=zeros(mx,1);
Index=1;
flag=0;
for i=1:m
for j=1:n
if L(i,j)~=0
for k=1:mx
if L(i,j)==L_number(k)
flag=1;
end
end
if flag~=1
L_number(Index)=L(i,j);
Index=Index+1;
end
flag=0;
end;
end
end
L_number;
Test_number=[3,4,6,7,8,9,10,11,14,15,19,20,21,22,24,26,27,28,29,30,31,32,33,35,
36,38,39,40,41,42,43,45,48,49,50,51,52,54,55,56,57,59,60,62,65,66];
for x=1:46
[r,c] = find(L==L_number((Test_number(x))));
rc = [r c];
[sx sy]=size(rc);
n1=zeros(imx,imy);
for i=1:sx
x1=rc(i,1);
y1=rc(i,2);
n1(x1,y1)=255;
end
%h=figure;imshow(n1,[]);
end
Circumference=zeros(46,1);
Arm_length=zeros(46,1);
Area=zeros(46,1);
for i=1:46
f=imread(strcat(num2str(i),'.bmp'));
BW=im2bw(f);
BW=double(BW);
BW1=edge(BW,'canny');
[m n]=size(BW1);
Circumference_sum=0;
for x=1:m
for y=1:n
if BW1(x,y)==1
Circumference_sum=Circumference_sum+1;
end
end
end
Circumference(i)=Circumference_sum;
f=imcomplement(f);
skel=im2double(f);
skel=im2bw(skel,1.5*graythresh(skel));
s=bwmorph(skel,'skel',Inf);
s1=bwmorph(s,'spur',8);
Arm_length_sum=0;
[m n]=size(s1);
for x=1:m
for y=1:n
if s1(x,y)==1
Arm_length_sum=Arm_length_sum+1;
end
end
end
Arm_length(i)=Arm_length_sum;
Area_sum=0;
BW=im2bw(f);
[m n]=size(BW);
for x=1:m
for y=1:n
if BW(x,y)==1
Area_sum=Area_sum+1;
end
end
end
Area(i)=Area_sum;
end
Circumference;
Arm_length;
Area;
Pair=zeros(46,2);
for i=1:45
min=abs(Circumference(i)-Circumference(i+1))+abs(Arm_length(i)-
Arm_length(i+1))+abs(Area(i)-Area(i+1));
Pair(i,1)=i;
Pair(i,2)=i+1;
for j=1:46
if i~=j && abs(Circumference(i)-Circumference(j))+abs(Arm_length(i)-
Arm_length(j))+abs(Area(i)-Area(j))<min
min=abs(Circumference(i)-Circumference(j))+abs(Arm_length(i)-
Arm_length(j))+abs(Area(i)-Area(j));
Pair(i,1)=i;
Pair(i,2)=j;
end
end
end
for i=1:45
if Pair(i,2)==46
Pair(46,1)=46;
Pair(46,2)=i;
end
end
Pair;
delete=zeros(46,1);
flag=0;
figure_flag=1;
for i=1:46
for j=1:46
if Pair(i,1)==delete(j)
flag=1;
end
end
if flag~=1
if figure_flag~=47
subplot(23,2,figure_flag);
figure_flag=figure_flag+1;
end
f1=imread(strcat(num2str(Pair(i,1)),'.bmp'));
imshow(f1);
if figure_flag~=47
subplot(23,2,figure_flag);
figure_flag=figure_flag+1;
end
f2=imread(strcat(num2str(Pair(i,2)),'.bmp'));
imshow(f2);
delete(figure_flag)=Pair(i,2);
end
flag=0;
end
CONCLUTION
In this paper, a newmetric is proposed to measure the distance
between chromosomes to be used in the automatic chromosome pairing procedure,
in the scope of karyotyping process used in cytogentic analysis. The proposed
algorithm is based on the traditional features extracted from the karyogram, such
as, dimensions and banding profiles, plus a new one, based on the MI, to improve
the discriminative power of the pairing algorithm with respect to the the G-banding
pattern. The main goal of this paper is to provide useful contributions toward the
design of a fully automatic chromosomes pairing algorithm of bone marrow cells
to be used in the diagnosis of leukemia. The images of these chromosomes present
less quality and level of detail than the ones usually used in traditional genetic
analysis using datasets such as Edinburgh, Copenhagen, and Philadelphia. The
algorithm is composed by four main steps: 1) image processing of the karyograms
provided by the technicians; 2) feature extraction from the processed images
characterizing the size, shape and band pattern; 3) training of a classifier
(performed once) where similarity among chromosomes are characterized; and
finally, 4) pairing. In the image processing step, the romosome images, extracted
from the unordered karyogram, are processed in order to compensate for
geometrical and intensity distortions, and to normalize their dimensions. This
normalization is needed to make it possible the band pattern comparison between
chromosomes. The features extracted from the processed images discriminate each
pair with respect to their size, shape, and band pattern. Here, a novel metric
distance is proposed to be used in the pairing procedure that linearly combines the
distances associated with each feature. The coefficients of the linear combination
are obtained through a training step using chromosomes paired manually by
experts. Vectors of coefficients associated with each one of the 22 classes are
computed and the distance between two arbitrary chromosomes is the minimum
one among all distances obtained with these 22 vectors. The training process
consists in the estimation of each vector of coefficient , from the chromosomes in
the training set, by minimizing the overall distances between chromosomes of the
same class (intraclass) and by maximizing the distances between chromosomes
when at least one of them does not belong to that class (interclass). The pairing
process is performed by efficiently solving a combinatorial problem where a
permutation matrix is obtained from the distance matrix computed with the
extracted features associated with each pair of chromosomes in the karyogram.
Tests using 19 karyograms based on bone marrow cells,working with 22 classes of
chromosomes and a LOOCV strategy allowus to conclude that the proposed
pairing algorithms,working within an 8-D feature space, achieves a 70.10% mean
classification rate. The addition of the MI feature to the traditional geometrical and
band profile features described in the literature leads to a clear improvement in the
performance of the classifier. Executing the algorithm on a higher quality dataset, a
76.10% classification ratewas obtained. Using 27 karyograms andworking with a
limited number of classes (≤ 8), amean classification rate larger than 93% was
obtained in all experiments. Qualitative comparisons with the results obtained with
the Leica CW 4000 Karyopairing software using the same data were performed
and have shown relevant improvements. In addition, a new chromosome dataset
with 9200 chromosomes from bone marrow cells, called LK1 , was built to provide
a ground truth to test classification and pairing algorithms for this type of “low”
image quality chromosomes. This dataset was made publicly available [29]. The
results presented in this paper are promising. In fact, despite the low quality of this
type of chromosomes, it was shown that it is possible to achieve comparable
classification rates to the ones obtained with the classical chromosome dataset,
such as Edinburgh, Copenhagen, or Philadelphia, whose images are of significantly
higher quality, presenting a uniform level of condensation, and from which it is
possible to extract additional features, e.g., centromere position.
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