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.
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 nucleotides in a long chain. Typically, eukaryotic cells (cells with nuclei) have large linear chromosomes andprokaryotic cells (cells without
The structure of chromosomes and chromatin varies through the cell cycle. circular DNA molecules called plasmids. These small circular genomes
. mitochondria in most eukaryotes and chloroplasts in plants have their own small chromosomes. through processes known as chromosomal instability and translocation. In prokaryotes and viruses.defined nuclei) have smaller circular chromosomes. Chromosomes are the essential unit for cellular division and must be replicated. Also. which is tightly coiled in on itself. cells may contain more than one type of chromosome. This allows the very long DNA molecules to fit into the cell nucleus. sometimes accompanied by one or more smaller. Chromosomal recombination plays a vital role in genetic diversity. Unduplicated chromosomes are single linear strands. If these structures are manipulated incorrectly. However. the cell may undergo mitotic catastrophe and die. or it may unexpectedly evadeapoptosis leading to the progression of cancer. DNA is usually arranged as a circle. for example. In practice "chromosome" is a rather loosely defined term. and passed successfully to their daughter cells so as to ensure the genetic diversity and survival of their progeny. divided. In prokaryotes. a large body of work uses the term chromosome regardless of chromatin content. although there are many exceptions to this rule. whereas duplicated chromosomes contain two identical copies (called chromatids) joined by a centromere. Chromosomes may exist as either duplicated or unduplicated. Compaction of the duplicated chromosomes during mitosis and meiosis results in the classic four-arm structure (pictured to the right). In eukaryotes. nuclear chromosomes are packaged by proteins into a condensed structure called chromatin. the term genophore is more appropriate when no chromatin is present.
in which an individual has 3. XX (female) or 46 XY (male).3 MUTATIONS IN CHROMOSOME NUMBER Normally. Euploid human karyotypes are 46. The simplest gonophores are found in viruses: these DNA or RNA molecules are short linear or circular gonophores that often lack structural proteins.
1. Such individuals are called euploid and have the wild-type chromosome complement for the species. rather than 2. If the mutation involves only one or a few chromosomes in the genome (e.+21.g. The individual would have Down Syndrome and his/her karyotype would be written 47.XY or 47.are also found in mitochondria and chloroplasts.XX.+21. reflecting their bacterial origins. the individual carrying the mutation is said to be aneuploid. copies of chromosome 21. a extra copy of human chromosome 21). 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. 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). An example of aneuploidy is trisomy 21.
They cease to function as accessible genetic material (transcription stops) and become a compact transportable form. a pair of sister chromatids attached to each other at the centromere. the chromatin strands become more and more condensed. This is the only natural context in which individual chromosomes are visible with an optical microscope. chromosomes are structurally highly condensed. 1. and they form the classic four arm structure. In spite of their appearance. Such a failure of the separation of homologous chromosomes or sister chromatids is called nondisjunction. q-g "grande"). which enables these giant DNA structures to be contained within a cell nucleus.1 chromosome paired model Aneuploidy is usually caused by spindle fiber failure in meiosis I or II. one of which is present on each sister chromatid. the chromatids are uncoiled and DNA can again be transcribed. longer-lasting attachment in this region.
. along with special proteins. This compact form makes the individual chromosomes visible. small) and the longer arms are called q arms (q follows p in the Latin alphabet. so that each daughter cell inherits one set of chromatids. Once the cells have divided. The microtubules then pull the chromatids apart toward the centrosomes. The shorter arms are called p arms (from the French petit.4 Metaphase chromatin and division In the early stages of mitosis or meiosis (cell division).Fig 1. microtubules grow from centrosomes located at opposite ends of the cell and also attach to the centromere at specialized structures called kinetochores. A special DNA base sequence in the region of the kinetochores provides. During mitosis.
7 lbs). which use the bone marrow vasculature as a conduit to the body's systemic circulation. in two separate nuclei. It is generally followed immediately by cytokinesis. cytoplasm. On average. which divides the nuclei.6 kg (5. bone marrow accounts for approximately 2. organelles and cell membrane into two cells containing roughly equal shares of these cellular components.5 BONE MARROW Bone marrow (Latin: medulla ossium) is the flexible tissue found in the interior of bones. in adults weighing 65 kg (143 lbs). bone marrow in large bones produces new blood cells.  Bone marrow is also a key component of the lymphatic system. Mitosis and cytokinesis together define the mitotic (M) phase of the cell cycle—the division of the mother cell into two daughter cells. In humans.1 MITOSIS Mitosis is the process by which a eukaryotic cell separates the chromosomes in its cell nucleus into two identical sets.
. bone marrow constitutes 4% of the total body mass of humans. producing the lymphocytes that support the body's immune system
CHAPTER 2 2. The hematopoietic compartment of bone marrow produces approximately 500 billion blood cells per day.1.
The sequence of events is divided into stages corresponding to the completion of one set of activities and the start of the next. where chromosomes divide within an intact cell nucleus. but is found in various different groups. During mitosis the pairs of chromatids condense and attach to fibers that pull the sister chromatids to opposite sides of the cell. Errors in mitosis can either kill a cell through apoptosis or cause mutations that may lead to cancer. Prokaryotic cells. This accounts for approximately 10% of the cell cycle. for instance during certain stages of fruit fly embryonic development. Even in animals. Because cytokinesis usually occurs in conjunction with mitosis.
. prometaphase. metaphase. which lack a nucleus. animals undergo an "open" mitosis. For example. The cell then divides in cytokinesis. there are many cells where mitosis and cytokinesis occur separately. anaphase and telophase. to produce two identical daughter cells which are still diploid cells. where the nuclear envelope breaks down before the chromosomes separate. This occurs most notably among the fungi and slime moulds. cytokinesis and mitosis may occur independently. while fungi such as Aspergillus nidulans and Saccharomyces cerevisiae (yeast) undergo a "closed" mitosis. These stages are interphase. prophase. Mitosis occurs only in eukaryotic cells and the process varies in different species.genetically identical to each other and to their parent cell. divide by a process called binary fission. forming single cells with multiple nuclei. The process of mitosis is fast and highly complex. "mitosis" is often used interchangeably with "mitotic phase". However.
the period that precedes the mitotic phase in the cell cycle where preparation for mitosis occurs.
. The sister chromatids are held together by a specialized region of the chromosome known as the centromere. Each chromosome now has an identical copy of itself. This occurs during the S phase of interphase.Fig 3 Mitosis cell division The primary result of mitosis is the transferring of the parent cell's genome into two daughter cells. The genome is composed of a number of chromosomes—complexes of tightly-coiled DNA that contain genetic information vital for proper cell function. the parent cell must make a copy of each chromosome before mitosis. These two cells are identical and do not differ in any way from the original parent cell. Because each resultant daughter cell should be genetically identical to the parent cell. and together the two are called sister chromatids.
As a matter of convention. the process of binary fission is very much different from the process of mitosis. pulling apart the sister chromatids of each chromosome. In plant cells. In animal cells. the nuclear envelope which segregates the DNA from the cytoplasm disassembles. the daughter cells will construct a new dividing cell wall between each other. As mitosis completes. the parent cell will be split in half. so they are renamed to sister chromosomes. separating the two developing nuclei.In most eukaryotes. Microtubules — essentially miniature strings— splay out from opposite ends of the cell and shorten. because of the non-involvement of nuclear dynamics and lack of linear chromosomes. each sister chromatid is now considered a chromosome. 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). each with a replica of the original genome. Prokaryotic cells undergo a process similar to mitosis called binary fission.
. corresponding sister chromosomes are pulled toward opposite ends. Eventually. As the cell elongates. A new nuclear envelope forms around the separated sister chromosomes.the cell begins cytokinesis. The chromosomes align themselves in a line spanning the cell. However. giving rise to two daughter cells.
However. 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. a transverse sheet of cytoplasm that bisects the cell along the future plane of cell
.1 Preprophase In plant cells only. 2. prophase is preceded by a pre-prophase stage. It alternates with the much longer interphase. Interphase is divided into three phases: G1 (first gap). grows more and prepares for mitosis (G 2). S (synthesis). During all three phases.2.2.2 Phases of cell cycle and mitosis Interphase
Fig 4 The cell cycle
The mitotic phase is a relatively short period of the cell cycle. the nucleus has to migrate into the center of the cell before mitosis can begin. This is achieved through the formation of a phragmosome. the cell grows by producing proteins and cytoplasmic organelles. All these phases in the interphase are highly regulated. In highly vacuolated plant cells. continues to grow as it duplicates its chromosomes (S). Thus. a cell grows (G1). chromosomes are replicated only during the S phase. and finally it divides (M) before restarting the cycle. mainly via proteins. where the cell prepares itself for cell division. and G2 (second gap).
Telophase: The decondensing chromosomes are surrounded by nuclear membranes.division.
. The preprophase band disappears during nuclear envelope disassembly and spindle formation in prometaphase. Cytokinesis has already begun. The chromosomes have chromatin has condensed. after the nuclear membrane breaks down. degraded.
Prophase: The two round objects above the nucleus Metaphase: The are the centrosomes. 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. aligned at the metaphase plate. This band marks the position where the cell will eventually divide. the pinched area is known as the cleavage furrow. microtubules form a spindle on the surface of the nucleus and are then organized into a spindle by the chromosomes themselves. In addition to phragmosome formation. and microtubules have invaded the nuclear Prophase space. instead.
Early anaphase: The Prometaphase: The kinetochore nuclear membrane has microtubules shorten. These microtubules can attach to kinetochores or they can interact with opposing microtubules. The cells of higher plants (such as the flowering plants) lack centrioles.
At the onset of prophase. chromatin condenses together into a highly ordered structure called a chromosome. The centrosome is the coordinating center for the cell's microtubules. Close to the nucleus are structures called centrosomes. which are made of a pair of centrioles found in most eukaryotic animal cells. the genetic material in the nucleus is in a loosely bundled coil called chromatin. they are not essential for the
. Molecular motor proteins then push the centrosomes along these microtubules to opposite sides of the cell.Fig 5 Micrograph showing condensed chromosomes in blue and the mitotic spindle in green during prometaphase of mitosis Normally. A cell inherits a single centrosome at cell division. The two centrosomes nucleate microtubules (which may be thought of as cellular ropes or poles) to form the spindle by polymerizing soluble tubulin. Since the genetic material has already been duplicated earlier in S phase. the replicated chromosomes have two sister chromatids. giving a pair of centrosomes. Although centrioles help organize microtubule assembly. which is replicated by the cell with the help of the nucleus before a new mitosis begins. Chromosomes are typically visible at high magnification through a light microscope. bound together at the centromere by the cohesin protein complex.
2. one attached at each chromatid. 2. since they are absent from plants.
. kinetochore microtubules begin searching for kinetochores to attach to. it is the point where microtubules attach themselves to the chromosome ( about 1-40 in number. on an average 20 ). and centrosomes are not always used in mitosis. Fungi and some protists. This is called open mitosis. undergo a variation called closed mitosis where the spindle forms inside the nucleus. the motor activates. When the spindle grows to sufficient length. Each chromosome forms two kinetochores at the centromere. it is known that it contains some form of molecular motor. Prometaphase is sometimes considered part of prophase. using energy from ATP to "crawl" up the tube toward the originating centrosome. A number of nonkinetochore microtubules find and interact with corresponding nonkinetochore microtubules from the opposite centrosome to form the mitotic spindle.formation of the spindle. coupled with polymerisation and depolymerisation of microtubules. This motor activity.2 Prometaphase The nuclear envelope disassembles and microtubules invade the nuclear space. or its microtubules are able to penetrate an intact nuclear envelope. Although the kinetochore structure and function are not fully understood. provides the pulling force necessary to later separate the chromosome's two chromatids. When a microtubule connects with the kinetochore. and it occurs in most multicellular organisms. such as algae or trichomonads. A kinetochore is a complex protein structure that is analogous to a ring for the microtubule hook.
Metaphase comes from the Greek meaning "after. an imaginary line that is equidistant from the two centrosome poles. As a result. convene along the metaphase plate or equatorial plane.In the fishing pole analogy.3 Metaphase
A cell in late metaphase. The centrosome acts as the "reel" that draws in the spindle fibers or "fishing line". In certain types of cells. the kinetochore would be the "hook" that catches a sister chromatid or "fish".
. only roughly lining up along the midline.
2. The centromeres of the chromosomes. the chromosomes come under longitudinal tension from the two ends of the cell." Microtubules find and attach to kinetochores in prometaphase. This even alignment is due to the counterbalance of the pulling powers generated by the opposing kinetochores. Then the two centrosomes start pulling the chromosomes through their attached centromeres towards the two ends of the cell. in some sense. It is also one of the main phases of mitosis because without it cytokinesis would not be able to occur. analogous to a tug-of-war between people of equal strength. All chromosomes (blue) but one have arrived at the metaphase plate. chromosomes do not line up at the metaphase plate and instead move back and forth between the poles randomly.
” “back. the proteins that bind sister chromatids together are cleaved. are pulled apart by shortening kinetochore microtubules and move toward the respective centrosomes to which they are attached. Two events then occur: first. allowing them to separate. These sister chromatids.Because proper chromosome separation requires that every kinetochore be attached to a bundle of microtubules (spindle fibres). The signal creates the mitotic spindle checkpoint. The force that causes the centrosomes to move towards the ends of the cell is still unknown.” “against.4 Anaphase When every kinetochore is attached to a cluster of microtubules and the chromosomes have lined up along the metaphase plate. Early anaphase is usually defined as the separation of the sister chromatids. pulling the centrosomes (and the set of chromosomes to which they are attached) apart to opposite ends of the cell. At the end of anaphase.” or “re-”). which have now become distinct sister chromosomes. it is thought that unattached kinetochores generate a signal to prevent premature progression to anaphase without all chromosomes being aligned. while late anaphase is the elongation of the microtubules and the chromosomes being pulled farther apart. the cell has succeeded in separating identical copies of the genetic material into two distinct populations. the nonkinetochore microtubules elongate. Next.
. 2. These two stages are sometimes called early and late anaphase. the cell proceeds to anaphase (from the Greek meaning “up. although there is a theory that suggests that the rapid assembly and breakdown of microtubules may cause this movement.
with the cleavage furrow being clearly visible
Cytokinesis is often mistakenly thought to be the final part of telophase. the nonkinetochore microtubules continue to lengthen. A new nuclear envelope. cell
. forms around each set of separated sister chromosomes. a cleavage furrow (pinch) containing a contractile ring develops where the metaphase plate used to be. In both animal and plant cells. now surrounded by new nuclei. however. Cytokinesis is technically not even a phase of mitosis. Mitosis is complete. At telophase. but rather a separate process. necessary for completing cell division.2. In animal cells. It "cleans up" the after effects of mitosis.5 Telophase Telophase (from the Greek meaning "end") is a reversal of prophase and prometaphase events. Both sets of chromosomes. cytokinesis is a separate process that begins at the same time as telophase.5 Cytokinesis
Cilliate undergoing cytokinesis. but cell division is not yet complete. pinching off the separated nuclei.
2. using fragments of the parent cell's nuclear membrane. Corresponding sister chromosomes attach at opposite ends of the cell. unfold back into chromatin. elongating the cell even more.
which move along microtubules to the middle of the cell. zygote and also the basis of the growth of a multicellular body. Following are the occasions in the lives of organism where mitosis happens: 2.3 Cell replacement In some parts of body.5. 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.e. RBCs have short life span (only about 4 months) and new RBCs are formed by mitosis.5. 2. cells are constantly sloughed off and replaced by new ones. e. whereas some green algae use a phycoplast microtubule array during cytokinesis. The phragmoplast is a microtubule structure typical for higher plants.g. 2. In plants this structure coalesces into a cell plate at the center of the phragmoplast and develops into a cell wall. This is the basis of the development of a multicellular body from a single cell i.4 Regeneration
.2 Development and growth The number of cells within an organism increases by mitosis.5. each cell formed receives chromosomes that are alike in composition and equal in number to the chromosomes of the parent cell. Similarly.1Significance Mitosis is important for the maintenance of the chromosomal set. skin and digestive tract.division is also driven by vesicles derived from the Golgi apparatus. separating the two nuclei.5.. New cells are formed by mitosis and so are exact copies of the cells being replaced. 2.
Occasionally when cells experience nondisjunction. The cells at the surface of hydra undergo mitosis and form a mass called bud. a condition often associated with cancer. For example. and the latter cell having only one chromosome (the homologous chromosome). In non-disjunction. the hydra reproduces asexually by budding.5.5. The production of new cells is achieved by mitosis.6 Asexual reproduction Some organisms produce genetically similar offspring through asexual reproduction. especially during early cellular divisions in the zygote. sea star regenerates its lost arm through mitosis. This results in the former cell having three chromosomes containing the same genes (two sisters and a homologue). 2. resulting in binucleated cells. a condition known as monosomy.Some organisms can regenerate their parts of bodies. a condition known as trisomy. the process may go wrong.7 Consequences of errors Although errors in mitosis are rare.
. a chromosome may fail to separate during anaphase. Mitosis continues in the cells of bud and it grows into a new individual. they fail to complete cell division and retain both nuclei in one cell. Mitotic errors can be especially dangerous to the organism because future offspring from this parent cell will carry the same disorder. For example. One daughter cell will receive both sister chromosomes and the other will receive none. The same division happens during asexual reproduction or vegetative propagation in plants. 2. These cells are considered aneuploid.
Now what happens is that cell abnormally continue to divide at a single place. it results in the formation of Tumors.Mitosis is a demanding process for the cell. causing deletion. which goes through dramatic changes in ultrastructure. and chromosomes are jostled constantly by probing microtubules. causing translocation. non-homologous chromosome. Malignant tumors are also known as cancerous tumours and their cells are called cancerous tumours. When tissues more than the requirement are synthesized in a single organ. causing chromosomal duplication. causing inversion. Or. It results in abnormal cell growth. Such tumours can send cancer cells to other parts in body where new tumours may form. chromosomes may become damaged. Occasionally. Errors in the control of mitosis may cause cancer. As long as these tumours remain in their original location they are called benign tumours. It may reattach to the original chromosome.
. it may be treated erroneously as a separate chromosome. Benign tumours are not harmful as soon as they are not moving. An arm of the chromosome may be broken and the fragment lost. but in reverse orientation. The effect of these genetic abnormalities depends on the specific nature of the error. As soon as they start to move and invade other cells there are said to be malignant tumours. This phenomenon is called metastasis or spreading of disease. All cells have genes that control the timing and number of mitosis. It results in the synthesis of execessive tissue growths. The fragment may incorrectly reattach to another. sometimes mutuations occur in such genes and cells continue to divide. its organelles disintegrate and reform in a matter of hours.
only roughly lining up along the middleline. an imaginary line that is equidistant from the two centrosome poles. align in the middle of the cell before being separated into each of the two daughter cells. This even alignment is due to the counterbalance of the pulling powers generated by the opposing kinetochores. is a stage of mitosis in the eukaryotic cell cycle in which condensed & highly coiled chromosomes. analogous to a tug of war between equally strong people. Early events of metaphase can
. The centromeres of the chromosomes convene themselves on the metaphase plate (or equatorial plate). In certain types of cells.7 Metaphase Metaphase. microtubules formed in prophase have already found and attached themselves to kinetochores in metaphase.2.6 Endomitosis Endomitosis is a variant of mitosis without nuclear or cellular division. Preceded by events in prometaphase and followed by anaphase. carrying genetic information. This process may also be referred to as endoreduplication and the cells as endoploid.
2. Metaphase accounts for approximately 4% of the cell cycle's duration. resulting in cells with many copies of the same chromosome occupying a single nucleus. from the ancient Greek(between) and (stage). chromosomes do not line up at the metaphase plate and instead move back and forth between the poles randomly. An example of a cell that goes through endomitosis is the megakaryocyte.
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. 2. Staining of the slides. even if most of the kinetochores have been attached and most of the chromosomes have been aligned. securin. One of the cell cycle checkpoints occurs during prometaphase and metaphase. Normal metaphase spreads are used in
. This would be accomplished by regulation of the anaphase-promoting complex. often with Giemsa (G banding) or Quinacrine. when every kinetochore is properly attached to a bundle of microtubules. cells are grown in short term culture and arrested in metaphase using mitotic inhibitor. Only after all chromosomes have become aligned at the metaphase plate. Metaphase chromosomes make the classical picture of chromosomes (karyotype). which makes them most suitable for visual analysis.coincide with the later events of prometaphase. produces a pattern of in total up to several hundred bands. For classical cytogenetic analyses. does the cell enter anaphase. Such a signal creates the mitotic spindle checkpoint. It is thought that unattached or improperly attached kinetochores generate a signal to prevent premature progression to anaphase. and separase.8 Metaphase in cytogenetics and cancer studies The analysis of metaphase chromosomes is one of the main tools of classical cytogenetics and cancer studies. 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). Chromosomes are condensed(Thickened) and highly coiled in metaphase.
which may lead to chimeric oncogenes.
. such as bcr-abl in chronic myelogenous leukemia. Inspection of the stained metaphase chromosomes allows the determination of numerical and structural changes in the tumor cell genome. for example.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. losses of chromosomal segments or translocations.
in humans 2n = 46. any differences between the sex chromosomes. to study chromosomal aberrations.CHAPTER 3 KARYOTYPING A karyotype is the number and appearance of chromosomes in the nucleus of an eukaryotic cell. cellular function. banding pattern. Thus. Karyotypes describe the number of chromosomes. in normal diploid organisms. The basic number of chromosomes in the somatic cells of an individual or a species is called the somatic number and is designated 2n. or an individual organism. In the germ-line (the sex cells) the chromosome number is n (humans: n = 23).  The preparation and study of karyotypes is part of cytogenetics. such as. The study of karyotypes is important for cell biology and genetics. and what they look like under a light microscope. and the results may be used in evolutionary biology and medicine. The term is also used for the complete set of chromosomes in a species. Attention is paid to their length. and to gather information about past evolutionary events. autosomal chromosomes are present in two copies. be sex chromosomes. or may not. the position of the centromeres. Karyotypes can be used for many purposes. taxonomic relationships. The study of whole sets of chromosomes is sometimes known as karyology. ordered by size and position of centromere for chromosomes of the same size. So. Karyogram of human male using Giemsa staining. There may. and any other physical characteristics. Polyploid cells have multiple copies of chromosomes and haploid cells have single copies.
. The chromosomes are depicted (by rearranging a microphotograph) in a standard format known as a karyogram or idiogram: in pairs.
at first favoring 46.
Pretreating cells in a hypotonic solution. which swells them and spreads the chromosomes
. The subsequent history of the concept can be followed in the works of Darlington and White. and he correctly insisted on humans having an XX/XY system. when it was appreciated that the set of chromosomes (the karyotype) was the carrier of the genes. the discoverer of mitosis. Their behavior in animal (salamander) cells was described by Walther Flemming. Levitsky seems to have been the first to define the karyotype as the phenotypic appearance of the somatic chromosomes. Using cells in culture
2. The name was coined by another German anatomist. The next stage took place after the development of genetics in the early 20th century. Painter in 1922 was not certain whether the diploid number of humans was 46 or 48. these results were quite remarkable. He revised his opinion later from 46 to 48. New techniques were needed to definitively solve the problem: 1. Hans von Winiwarter reported 47 chromosomes in spermatogonia and 48 in oogonia.1 History of karyotype studies Chromosomes were first observed in plant cells by Karl Wilhelm von Nägeli in 1842. Considering their techniques. 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. von Waldeyer in 1888. in contrast to their genic contents. in 1882. concluding an XX/XO sex determination mechanism.3.
Cutting up a photomicrograph and arranging the result into an indisputable karyogram.
3.1 Staining The study of karyotypes is made possible by staining. Squashing the preparation on the slide forcing the chromosomes into a single plane 5. The
sex of an unborn fetus can be determined by observation of interphase cells.2 Observations Six different characteristics of karyotypes are usually observed and compared:
. Rather interestingly. 3.
Sometimes observations may be made on non-dividing (interphase) cells. reducing the number. a suitable dye. such as Giemsa. Usually. For humans.2. is applied after cells have been arrested during cell division by a solution of colchicine. the great apes have 48 chromosomes. Human chromosome 2 was formed by a merger of ancestral chromosomes. white blood cells are used most frequently because they are easily induced to divide and grow in tissue culture.3.2 Observations on karyotypes 3.
Arresting mitosis in metaphase by a solution of colchicines
4. It took until the mid 1950s until it became generally accepted that the karyotype of humans included only 46 chromosomes.2.
Differences in absolute sizes of chromosomes. Differences in number and position of satellites. both have six pairs of chromosomes (n=6) yet V. Differences in basic number of chromosomes may occur due to successive unequal translocations which finally remove all the essential genetic material from a chromosome. Differences in relative size of chromosomes can only be caused by segmental interchange of unequal lengths.
Heterochromatin stains darker than euchromatin.
6. type. and mainly consists of genetically inactive repetitive DNA sequences. Differences in the position of centromeres.
. which (when they occur) are small bodies attached to a chromosome by a thin thread. 5. Chromosomes can vary in absolute size by as much as twenty-fold between genera of the same family: Lotus tenuis and Vicia faba (legumes).
of heterochromatic regions. This feature probably reflects different amounts of DNA duplication. shape and banding of the chromosomes. but the genes have been mostly translocated (added) to other chromosomes. faba chromosomes are many times larger. This is brought about by translocations. as well as other cytogenetic information. A full account of a karyotype may therefore include the number. 4. Humans have one pair fewer chromosomes than the great apes.1. indicating tighter packing. permitting its loss without penalty to the organism (the dislocation hypothesis).
3 The human karyotype Most (but not all) species have a standard karyotype. which are highly variable. Between the sexes
2. Between members of a population (chromosome polymorphism) 4.
Between the germ-line and soma (between gametes and the rest of the body)
3. The normal human karyotypes contain 22 pairs of autosomal chromosomes and one pair of sex chromosomes. Any variation from the standard karyotype may lead to developmental abnormalities. XX. Mosaics or otherwise abnormal individuals. There is variation between species in chromosome number. Geographical variation between races 5. Normal karyotypes for females contain two X chromosomes and are denoted 46.
Fig Diversity and evolution of karyotype Although the replication and transcription of DNA is highly standardized in eukaryotes.Variation is often found: 1. males have both an X and a Y chromosome denoted 46.
3. the same cannot be said for their karyotypes. and in
despite their construction from the same macromolecules. the general significance of karyotype evolution is obscure.. "We have a very poor understanding of the causes of karyotype evolution. as in many sciarid flies. This variation provides the basis for a range of studies in evolutionary cytology.3. In some cases there is even significant variation within species.. In a review.. In A. This process is a carefully organised genome rearrangement where new telomeres are constructed and certain heterochromatin regions are lost. karyotypic fissioning may help to explain dramatic differences in diploid numbers between closely related species. In this process. entire chromosomes are eliminated during development. found in some copepods and roundworms such as Ascaris suum.1 Changes during development Instead of the usual gene repression. 3. In some species. portions of the chromosomes are cast away in particular cells. Godfrey and Masters conclude: "In our view. 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. used in conjunction with other phylogenetic data. But. or other kinds of visible adjustment to the karyotype. some organisms go in for large-scale elimination of heterochromatin.detailed organization. it is unlikely that one process or the other can independently account for the wide range of karyotype structures that are observed. Although much is known about karyotypes at the descriptive level.. despite many careful investigations. Chromosome elimination.
. which were previously inexplicable. Chromatin diminution (founding father: Theodor Boveri).
In human females some 15% of somatic cells escape inactivation. In marsupials it is always the paternal X which is inactivated. the high record would be somewhere amongst the ferns. Xinactivation. "They simply could not believe what they saw. The diploid number of the Chinese muntjac. The existence of supernumerary or B chromosomes
. Muntiacus muntjak.. all the somatic cell precursors undergo chromatin diminution. the inactivation is random as between the two Xs. The inactivation of one X chromosome takes place during the early development of mammals (see Barr body and dosage compensation). In placental mammals... Top score for animals might be the shortnose sturgeon Acipenser brevirostrum at a mere 372 chromosomes. thus the mammalian female is a mosaic in respect of her X chromosomes. they were astonished to find it had female = 6. The low record is held by the nematode Parascaris univalens. all telocentric. which was investigated by Kurt Benirschke and his colleague Doris Wurster. was found to be 46.. 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. male = 7 chromosomes. 3. They kept quiet for two or three years because they thought something was wrong with their tissue culture.3. When they looked at the karyotype of the closely related Indian muntjac. Muntiacus reevesi. where the haploid n = 1.2 Number of chromosomes in a set A spectacular example of variability between closely related species is the muntjac.suum. with the Adder's Tongue Fern Ophioglossum ahead with an average of 1262 chromosomes.
Thus. and the other haploid.4 Ploidy
Ploidy is the number of complete sets of chromosomes in a cell. FN. FN ≤ 2n. 21 and 22). about 70%. though in this case they would not be regarded as normal members of the population. where one sex is diploid. The proportion of flowering plants which are polyploid was estimated by Stebbins to be 30-35%. Polyploidy in lower plants (ferns. Polyploidy in animals is much less common.
3. Polyploidy. 15. 14. and aneuploids are another example.3.3 Fundamental number The fundamental number. 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. 3. due to the presence of five acrocentric chromosome pairs (13. but in grasses the average is much higher.Endopolyploidy occurs when in adult differentiated
. where there are more than two sets of homologous chromosomes in the cells. of a karyotype is the number of visible major chromosomal arms per set of chromosomes. the difference depending on the number of chromosomes considered single-armed (acrocentric or telocentric) present. It is a common arrangement in the Hymenoptera.means that chromosome number can vary even within one interbreeding population. and in some other groups. Haplo-diploidy. occurs mainly in plants. It has been of major significance in plant evolution according to Stebbins. 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. Humans have FN = 82.
and serves differentiation and morphogenesis in many ways. the daughter chromosomes separating from each other inside an intact nuclear membrane. Abnormalities in chromosome number usually cause a defect in development.
. The cells do not always contain exact multiples (powers of two). 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. Down syndrome and Turner syndrome are examples of this.5 Aneuploidy Aneuploidy is the condition in which the chromosome number in the cells is not the typical number for the species. In many instances. In the endocycle (endomitosis or endoreduplication) chromosomes in a 'resting' nucleus undergo reduplication. The phenomenon occurs sporadically throughout the eukaryote kingdom from protozoa to man. See palaeopolyploidy for the investigation of ancient karyotype duplications. which is why the simple definition 'an increase in the number of chromosome sets caused by replication without cell division' is not quite accurate. 3. it is diverse and complex. but the nuclei contain more than the original somatic number of chromosomes.tissues the cells have ceased to divide by mitosis. endopolyploid nuclei contain tens of thousands of chromosomes (which cannot be exactly counted). This would give rise to a chromosome abnormality such as an extra chromosome or one or more chromosomes lost.
In about 6.Aneuploidy may also occur within a group of closely related species. Evidence of various kinds shows that that trends of evolution have gone in different directions in different groups. 4. Classic examples in plants are the genus Crepis.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. 3. 5. the Hawaiian Islands have the most diverse collection of drosophilid flies in the world. reducing the number.500 sq mi (17. Well-researched examples are the ladybird beetle Chilocorus stigma. and Crocus.000 km2). where the gametic (= haploid) numbers form the series x = 3. living from rainforests to
. 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. the chromosome number is variable from one individual to another. When this happens. and 7.  Closer to home. where every number from x = 3 to x = 15 is represented by at least one species. 3. 6. that the two chromosome morphs are adapted to different habitats. the great apes have 24x2 chromosomes whereas humans have 23x2.5 Chromosomal polymorphism Some animal species are polymorphic for chromosome fusions or dissociations. Human chromosome 2 was formed by a merger of ancestral chromosomes.
the best-studied group of Hawaiian drosophilids. gene arrangements are visible in the banding patterns of each chromosome. make it possible to see which species are closely related. These roughly 800 Hawaiian drosophilid species are usually assigned to two genera. enabled Carson to work out the evolutionary tree long before genome analysis was practicable. The subsequent adaptive radiation was spurred by a lack of competition and a wide variety of niches. and skipping of islands. The inversions. In a sense. Chromosome rearrangements. probably 20 million years ago. especially inversions. The polytene banding of the 'picture wing' group.subalpine meadows. Drosophila and Scaptomyza. The archipelago itself (produced by the Pacific plate moving over a hot spot) has existed for far longer. at least into the Cretaceous. The results are clear. 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. the present islands date from 0. The oldest member of the Hawaiian archipelago still above the sea is Kure Atoll. 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. when plotted in tree form (and independent of all other information). show a clear "flow" of species from older to newer islands.
. but these are much less frequent. which can be dated to 30 mya.4 million years ago (mya) (Mauna Kea) to 10mya (Necker). Using K-Ar dating. in the family Drosophilidae. There are also cases of colonization back to older islands.
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 . human genome.7 Depiction of karyotypes 3. if less spectacular.7. The pattern of bands is very similar to that seen in G-banding. This method will normally produce 300-400 bands in a normal. The light regions tend to be euchromatic.
Q-banding is a fluorescent pattern obtained using quinacrine for staining.
. early-replicating and GC rich.
C-banding: Giemsa binds to constitutive heterochromatin.
3. R-banding is the reverse of G-banding (the R stands for "reverse").
T-banding: visualize telomeres.the dark regions tend to be heterochromatic. adaptive radiations. late-replicating and AT rich.There are other animals and plants on the Hawaiian archipelago which have undergone similar. so it stains centromeres. The dark regions are euchromatic (guanine-cytosine rich regions) and the bright regions are heterochromatic (thymine-adenine rich regions).
Karyotypes are arranged with the short arm of the chromosome on top. often Giemsa (G-banding). Giemsa is specific for the phosphate groups of DNA.2 Classic karyotype cytogenetics
Karyogram from a human female lymphocyte probed for the Alu sequence using FISH.7. respectively.
3. is used to stain bands on the chromosomes. the differently stained regions and sub-regions are given numerical designations from proximal to distal on the chromosome arms. both chromosomes in a pair will have the same banding pattern. In addition.•
Silver staining: Silver nitrate stains the nucleolar organization regionassociated protein. For example. In the "classic" (depicted) karyotype. Cri du chat syndrome involves a deletion on the short arm of
. Quinacrine binds to the adeninethymine-rich regions. This yields a dark region where the silver is deposited. denoting the activity of rRNA genes within the NOR. and the long arm on the bottom. less frequently Quinacrine. Some karyotypes call the short and long arms p and q. Each chromosome has a characteristic banding pattern that helps to identify them. a dye.
chromosome 5. which is written as 46. Because there are a limited number of spectrally-distinct fluorophores.del(5)(p15. Short sequences of DNA from specific loci all over the genome are isolated and enumerated. Fluorescently labeled probes for each chromosome are made by labeling chromosome-specific DNA with different fluorophores.XX.2) 3. 3. allowing the visualization of the individually colored chromosomes.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. Image processing software then assigns a pseudo color to each spectrally different combination. 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. This method is also known as virtual karyotyping.8 Digital karyotyping Digital karyotyping is a technique used to quantify the DNA copy number on a genomic scale.7.2. Spectral differences generated by combinatorial labeling are captured and analyzed by using an interferometer attached to a fluorescence microscope. The critical region for this syndrome is deletion of 15. It is written as 46. a combinatorial labeling method is used to generate many different colors.
X or 45. Both types of abnormalities can occur in gametes and therefore will be present in all cells of an affected person's body. the most common male chromosomal disease. Structural abnormalities often arise from errors in homologous recombination. often occur as a result of nondisjunction during meiosis in the formation of a gamete. or they can occur during mitosis and give rise to a genetic mosaic individual who has some normal and some abnormal cells. large-scale deletions or duplications. X0). or structural.
Edwards syndrome is caused by trisomy (three copies) of chromosome 18. although they generally do not survive to birth. a common chromosomal disease.1 CHROMOSOMAL ABNORMALITIES Chromosome abnormalities can be numerical. inversions. is caused by trisomy of chromosome 21. translocations.
Some disorders arise from loss of just a piece of one chromosome. Klinefelter syndrome. Chromosomal abnormalities that lead to disease in humans include
Turner syndrome results from a single X chromosome (45.
Patau syndrome is caused by trisomy of chromosome 13. including
. Down syndrome. Numerical abnormalities. trisomy 9 and trisomy 16. in which three copies of a chromosome are present instead of the usual two.4. otherwise known as 47. XXY is caused by an extra X chromosome. Also documented are trisomy 8. are common numerical abnormalities. as in the presence of extra or missing chromosomes. trisomies. also known as aneuploidy. as in derivative chromosome.
A chromosome anomaly. numerical and structural anomalies. abnormality or aberration reflects an atypical number of chromosomes or a structural abnormality in one or more chromosomes.
Angelman syndrome – 50% of cases have a segment of the long arm of chromosome 15 missing. example of imprinting disorder. Chromosome anomalies usually occur when there is an error in cell division following meiosis or mitosis. 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.
Chromosomal abnormalities can also occur in cancerous cells of an otherwise genetically normal individual. a deletion of the paternal genes. They can be organized into two basic groups. The name comes from the babies' distinctive cry.
Prader-Willi syndrome – 50% of cases have a segment of the long arm of chromosome 15 missing. There are many types of chromosome anomalies. a deletion of the maternal genes. from the loss of part of the short arm of chromosome 1. example of imprinting disorder. one well-documented example is the Philadelphia chromosome. 1p36 Deletion syndrome. a translocation mutation commonly associated with chronic myelogenous leukemia and less often with acute lymphoblastic leukemia.•
Cri du chat (cry of the cat).
. A chromosome anomaly may be detected or confirmed in this manner. caused by abnormal formation of the larynx. from a truncated short arm on chromosome 5.
4. Known disorders in humans include Wolf-Hirschhorn syndrome.3 Structural abnormalities When the chromosome's structure is altered. also known as Trisomy 21 (an individual with Down Syndrome has three copies of chromosome 21.). resulting in extra genetic material. There are two main types of translocations.2 Numerical Disorders This is called Aneuploidy (an abnormal number of chromosomes). rather than two). also called the terminal 11q deletion disorder. This can take several forms:
Deletions: A portion of the chromosome is missing or deleted. and occurs when an individual is missing either a chromosome from a pair (monosomy) or has more than two chromosomes of a pair (Trisomy. In a Robertsonian translocation. etc. Tetrasomy. 4. Duplications: A portion of the chromosome is duplicated.
Translocations: When a portion of one chromosome is transferred to another chromosome. In a reciprocal translocation. and Jacobsen syndrome. segments from two different chromosomes have been exchanged. which is caused by partial deletion of the short arm of chromosome 4. an entire chromosome has
. In humans an example of a condition caused by a numerical anomaly is Down Syndrome. Turner Syndrome is an example of a monosomy where the individual is born with only one sex chromosome. an X. 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.
the anomaly is present in every cell of the body. Therefore. 21 and 22. They often lead to an increased tendency to develop certain types of malignancies. 15. This can happen with or without loss of genetic material. 14.3 Inheritance Most chromosome abnormalities occur as an accident in the egg or sperm. This is why chromosome studies are often performed on parents when a child is found to have an anomaly. turned upside down and reattached.
Isochromosome: Formed by the mirror image copy of a chromosome segment including the centromere. therefore the genetic material is inverted. 4. and are therefore initially not inherited.
Inversions: A portion of the chromosome has broken off. can happen after conception. Chromosome instability syndromes are a group of disorders characterized by
chromosomal instability and breakage.in humans these only occur with chromosomes 13. Some anomalies.4 Cytogenetics Cytogenetics is a branch of genetics that is concerned with the study of the structure and function of the cell. other cytogenetic banding techniques.attached to another at the Centromere . as well
. resulting in Mosaicism (where some cells have the anomaly and some do not). It includes routine analysis of G-Banded chromosomes. however. 4.
Rings: A portion of a chromosome has broken off and formed a circle or ring. Chromosome anomalies can be inherited from a parent or be "de novo". especially the chromosomes.
which swells them and spreads the chromosomes
. von Waldeyer in 1888. these results were quite remarkable. concluding an XX/XO sex determination mechanism. Painter in 1922 was not certain whether the diploid number of man was 46 or 48.
Pre-treating cells in a hypotonic solution.5 Early years Chromosomes were first observed in plant cells by Karl Wilhelm von Nägeli in 1842. The name was coined by another German anatomist. Hans von Winiwarter reported 47 chromosomes in spermatogonia and 48 in oogonia. Levitsky seems to have been the first to define the karyotype as the phenotypic appearance of the somatic chromosomes. and he correctly insisted on man having an XX/XY system. 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. Considering their techniques. The next stage took place after the development of genetics in the early 20th century. He revised his opinion later from 46 to 48. when it was appreciated that the set of chromosomes (the karyotype) was the carrier of the genes.as molecular cytogenetics such as fluorescent in situ hybridization (FISH) and comparative genomic hybridization (CGH). in contrast to their genic contents. at first favoring 46. New techniques were needed to definitively solve the problem: 1. in 1882. Using cells in culture
2. Their behavior in animal (salamander) cells was described by Walther Flemming. 4. the discoverer of mitosis.
a find which eventually led to her Nobel Prize in 1983. McClintock continued her career in cytogenetics studying the mechanics and inheritance of broken and ring (circular) chromosomes of maize. 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. Dobzhansky and his co-workers collected Drosophila pseudoobscura and D. Rather interestingly. the great apes have 48 chromosomes.
Arresting mitosis in metaphase by a solution of colchicine
4. 4. reducing the number. 4.1 McClintock's work on maize Barbara McClintock began her career as a maize cytogeneticist.6.3. Human chromosome 2 was formed by a merger of ancestral chromosomes.2 Natural populations of Drosophila In the 1930s.6 Applications in biology 4.6. During her cytogenetic work. It took until 1956 until it became generally accepted that the karyotype of man included only 46 chromosomes. McClintock and Harriet Creighton demonstrated that cytological recombination of marked chromosomes correlated with recombination of genetic traits (genes). McClintock discovered transposons. In 1931. persimilis from wild populations in California and neighboring states. Using Painter's technique they studied the polytene
breeding and sampling whilst preventing escape. All the flies look alike whatever inversions they carry: this is an example of a cryptic polymorphism. 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. Lejeune discovered patients with Down syndrome had an extra copy of chromosome 21. which enabled feeding. In some congenital disorders.
. such as Down's syndrome. Down syndrome is also referred to as trisomy 21. This had the benefit of eliminating migration as a possible explanation of the results. In 1959. as they would if selectively neutral. Stocks containing inversions at a known initial frequency can be maintained in controlled conditions. 4. Using a method invented by L'Heretier and Teissier. but adjust to certain frequencies at which they become stabilised. 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.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. Dobzhansky bred populations in population cages. Evidence rapidly accumulated to show that natural selection was responsible. It was found that the various chromosome types do not fluctuate at random.chromosomes and discovered that the wild populations were polymorphic for chromosomal inversions. as with most polymorphisms.
In 1960. resulting in 47 total chromosomes. Peter Nowell and David Hungerford  discovered a small chromosome in the white blood cells of patients with Chronic myelogenous leukemia (CML). which is required in normal females to compensate for having two copies of the chromosome.Other numerical abnormalities discovered include sex chromosome abnormalities. the abnormal chromosome was shown by Janet Rowley to be the result of a translocation of chromosomes 9 and 22. in addition to other tests. with the development of more advanced techniques. an additional X chromosome in a male. This abnormal chromosome was dubbed the Philadelphia chromosome . and XXXX.as both scientists were doing their research in Philadelphia. XYY. The ability for mammals to tolerate aneuploidies in the sex chromosomes arises from the ability to inactivate them. is used today as a diagnostic for CML. Many other sex chromosome combinations are compatible with live birth including XXX. Thirteen years later. has Klinefelter's Syndrome. Trisomy 13 was associated with Patau's Syndrome and trisomy 18 with Edward's Syndrome.
. Pennsylvania. Identification of the Philadelphia chromosome by cytogenetics. which is why there is a phenotypic effect seen in individuals with extra X chromosomes. An individual with only one sex chromosome (the X) has Turner syndrome. Not all genes on the X Chromosome are inactivated.
FIG Advent of banding techniques
In the late 1960s. Caspersson developed banding techniques which differentially stain chromosomes. 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.8 Beginnings of molecular cytogenetics
. Techniques were expanded to allow for culture of free amniocytes recovered from amniotic fluid. Deletions within one chromosome could also now be more specifically named and understood. Deletion syndromes such as DiGeorge syndrome. Diagrams identifying the chromosomes based on the banding patterns are known as cytogenetic maps. This allows chromosomes of otherwise equal size to be differentiated as well as to elucidate the breakpoints and constituent chromosomes involved in chromosome translocations. and elongation techniques for all culture types that allow for higher resolution banding. Prader-Willi syndrome and others were discovered to be caused by deletions in chromosome material. 4.
. 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). Further advances in micromanipulation and examination of chromosomes led to the technique of chromosome microdissection whereby aberrations in chromosomal structure could be isolated.In the 1980s. cloned and studied in ever greater detail. While radioisotope-labeled probes had been hybridized with DNA since 1969. This change significantly increased the usage of probing techniques as fluorescent labeled probes are safer and can be used almost indefinitely.
CHAPTER 5 Techniques 5. advances were made in molecular cytogenetics.
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 (Rbanding) 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-2phenylindole (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,
and FORTRAN. such as C. Add-on toolboxes (collections of special-purpose MATLAB functions) extend the MATLAB environment to solve particular classes of problems in these application areas. CGH and Single nucleotide polymorphism-arrays.generally between 200 and 1000 cells are counted and scored. you can solve technical computing problems faster than with traditional programming languages. including signal and image processing. data analysis.
MATLAB MATLAB is a high-level technical computing language and interactive environment for algorithm development. data visualization. such as comparative genomic hybridization arrays.
. 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. Using MATLAB. financial modeling and analysis. For congenital problems usually 20 metaphase cells are scored. and numerical computation. You can use MATLAB in a wide range of applications. communications. and computational biology. control design. test and measurement. C++.
specifying data types. one line of MATLAB code can often replace several lines of C or C++ code. and allocating memory. The MATLAB language supports the vector and matrix operations that are fundamental to engineering and scientific problems. These effects must be compensated to improve the results of the pairing algorithm.
6. you can program and develop algorithms faster than with traditional languages because you do not need to perform lowlevel administrative tasks. In many cases. such as declaring variables. You can integrate your MATLAB code with other languages and applications. MATLAB eliminates the need for ‘for’ loops. It enables fast development and execution.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.
.MATLAB provides a number of features for documenting and sharing your work. With the MATLAB language. The image processing step is composed of the following operations. 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. As a result. and distribute your MATLAB algorithms and applications.
To compare chromosomes from a band pattern point of view. 4) Intensity compensation—The metaphase plaque from which the chromosomes are extracted does not present a uniform brightness and contrast.1) Chromosome extraction—Each chromosome is isolated from the unordered karyogram. or at least attenuated.
6. performed by using the algorithm is needed to obtain chromosomes with vertical medial axis. 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. geometrical and dimensional differences must be removed. Therefore.2 Concepts used in this phase
1) Image conversion 2) Denoising
. 2) Geometrical compensation—The geometric compensation. 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). the spatially scaled images are histogram equalized. To compensate for this inhomogeneity.
I). and the results might not be meaningful.3) Edge detection
Two dimensional convolutions. MATLAB filters the intensity values in the image. there are other functions that return a different image type as part of the operation they perform. green. you must first convert it to true color format.
6. and blue planes.2. the region of interest functions returns a binary image that you can use to mask an image for filtering or for other operations. If you attempt to filter the indexed image. RGB = cat (3. You can perform certain conversions just using MATLAB syntax. When you apply the filter to the true color image.
. For example. The resulting true color image has identical matrices for the red. so the image displays as shades of gray. as is appropriate.I. you can convert a grayscale image to true color format by concatenating three copies of the original matrix along the third dimension. listed in the following table. For example. MATLAB simply applies the filter to the indices in the indexed image matrix.1 Image conversion The toolbox includes many functions that you can use to convert an image from one type to another. if you want to filter a color image that is stored as an indexed image. In addition to these image type conversion functions. For example.I.
Usually we know what type of errors to expect. via satellite or wireless transmission. If one of these matrices describes a two-dimensional finite impulse response
.B) computes the two-dimensional convolution of matrices
and B. and we shall look at some of the more straightforward of them. ) Where the parameters available depend on the method used 6. If an image is being sent electronically from one place to another.2. Cleaning an image corrupted by noise is thus an important area of image restoration. hence we can choose the most appropriate method for reducing the effects.2.4 Denoising
We may define noise to be any degradation in the image signal. There is a large number of edge finding algorithms in existence. and hence the type of noise on the image.3 Two dimensional convolutions C = conv2(A.6. We may use edges to measure the size of objects in an image. we may expect errors to occur in the image signal. . caused by external disturbance. to recognize or classify objects.'method'.
6. These errors will appear on the image output in different ways depending on the type of disturbance in the signal. or through networked cable. .parameters.5 Edge detection Edges contain some of the most useful information in an image. The general Matlab command for finding edges is edge(image. to isolate particular objects from their background.
C = conv2(.(FIR) filter.[3 3])..
subsection of the two-dimensional convolution. If hcol is a column vector and hrow is a row vector.'sobel').nb]. rgb2gray im2bw(im. imedfilt2(im1.A)
dimension is equal to the sum of the corresponding dimensions of the input
is [ma. That is. minus one.bmp'). edge(im1.
first with the vector
along the rows and then returns a
with the vector hrow along the columns.A). this case is the same as
C = conv2(hcol*hrow. as specified by the shape parameter
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.na] and the size of
nb]+1)/2).na+nb-1]. the other matrix is filtered in two dimensions. The size of matrices. if the size of then the size of C is [ma+mb-1. The indices of the center element of B are defined as floor(([mb
C = conv2(hcol.
y1=rc(i. for i=1:m for j=1:n if L(i.j)~=0 for k=1:mx if L(i. mx=max(max(L)).[imx. L_number=zeros(mx.y1)=255. bwlabel(B.j)==L_number(k) flag=1. Msk conv2(double(BW).2). Index=1. [sx sy]=size(rc).
.1). n1(x1. [r.imy]=size(BW). nzeros(imx.n]=size(L).imy). rc = [r c].8). flag=0.c] = find(L==22). for i=1:sx x1=rc(i.1). MODULE 2 clc [m.double(msk)).
31.2).10.7. end flag=0.48.4.45.60. 36.c] = find(L==L_number((Test_number(x)))). [sx sy]=size(rc).52. end %h=figure.20. end.30.imshow(n1.
.59.9. Index=Index+22.214.171.124.38.1).35.66]. end end L_number.21.41.42. rc = [r c].).126.96.36.199.6.39.j).54. y1=rc(i. n1=zeros(imx.imy).65.51. for i=1:sx x1=rc(i. n1(x188.8.131.52.y1)=255.49.28.end end if flag~=1 L_number(Index)=L(i.15.19. Test_number=[3.11. for x=1:46 [r.184.108.40.206.56.55.
s1=bwmorph(s. Circumference_sum=0. BW1=edge(BW.'skel'. for x=1:m for y=1:n if BW1(x.'canny').1.'spur'.1). Area=zeros(46. for i=1:46 f=imread(strcat(num2str(i). Arm_length=zeros(46.y)==1 Circumference_sum=Circumference_sum+1. end end end Circumference(i)=Circumference_sum.1). skel=im2bw(skel. s=bwmorph(skel. BW=im2bw(f).8). skel=im2double(f).1). f=imcomplement(f). [m n]=size(BW1).bmp')).end Circumference=zeros(46. BW=double(BW).'. Arm_length_sum=0.
BW=im2bw(f). end Circumference.[m n]=size(s1). for x=1:m for y=1:n if s1(x.
. for x=1:m for y=1:n if BW(x.y)==1 Arm_length_sum=Arm_length_sum+1. end end end Arm_length(i)=Arm_length_sum. end end end Area(i)=Area_sum.y)==1 Area_sum=Area_sum+1. Area_sum=0. [m n]=size(BW). Arm_length.
Pair(i. Pair(i. 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. Pair(46.1)=i.
.Area.2).2)=i+1. Pair(i. end end end for i=1:45 if Pair(i.2)=j.1)=i. end end Pair.1)=46. 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)).2)==46 Pair(46.2)=i. Pair=zeros(46.
figure_flag=1.1)==delete(j) flag=1. end f2=imread(strcat(num2str(Pair(i.figure_flag).delete=zeros(46. figure_flag=figure_flag+1.'.bmp')).
.2)).2).'.2. end flag=0. delete(figure_flag)=Pair(i.2. imshow(f1). end end if flag~=1 if figure_flag~=47 subplot(23. if figure_flag~=47 subplot(23.1). figure_flag=figure_flag+1.1)). for i=1:46 for j=1:46 if Pair(i.figure_flag). end f1=imread(strcat(num2str(Pair(i.bmp')). flag=0. imshow(f2).
plus a new one. such as. 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. and Philadelphia. based on the MI. Copenhagen. 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. in the scope of karyotyping process used in cytogentic analysis.end
CONCLUTION In this paper. The algorithm is composed by four main steps: 1) image processing of the karyograms provided by the technicians. a newmetric is proposed to measure the distance between chromosomes to be used in the automatic chromosome pairing procedure. dimensions and banding profiles. The proposed algorithm is based on the traditional features extracted from the karyogram. 2) feature extraction from the processed images
characterizing the size. 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. 3) training of a classifier (performed once) where similarity among chromosomes are characterized. The training process consists in the estimation of each vector of coefficient . Here. shape and band pattern. from the chromosomes in the training set. achieves a 70. 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
. The coefficients of the linear combination are obtained through a training step using chromosomes paired manually by experts. and band pattern. extracted from the unordered karyogram. 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). 4) pairing. shape. 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. Tests using 19 karyograms based on bone marrow cells.10% mean classification rate. the romosome images. a novel metric distance is proposed to be used in the pairing procedure that linearly combines the distances associated with each feature.working with 22 classes of chromosomes and a LOOCV strategy allowus to conclude that the proposed pairing algorithms. In the image processing step.working within an 8-D feature space. 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. and finally.
called LK1 . Executing the algorithm on a higher quality dataset. whose images are of significantly higher quality. such as Edinburgh.10% classification ratewas obtained. a new chromosome dataset with 9200 chromosomes from bone marrow cells. Using 27 karyograms andworking with a limited number of classes (≤ 8). centromere position. and from which it is possible to extract additional features. Copenhagen. presenting a uniform level of condensation.performance of the classifier. Qualitative comparisons with the results obtained with the Leica CW 4000 Karyopairing software using the same data were performed and have shown relevant improvements. it was shown that it is possible to achieve comparable classification rates to the ones obtained with the classical chromosome dataset. e. amean classification rate larger than 93% was obtained in all experiments.g. The results presented in this paper are promising.
. despite the low quality of this type of chromosomes. was built to provide a ground truth to test classification and pairing algorithms for this type of “low” image quality chromosomes. In fact. or Philadelphia. This dataset was made publicly available . a 76.. In addition.
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